Method to determine responsiveness of cancer to epidermal growth factor receptor targeting treatments

ABSTRACT

The present invention is directed to a method for determining the responsiveness of cancer to an epidermal growth factor receptor (EGFR) treatment. In a preferred embodiment, the presence of at least one variance in the kinase domain of the erbB1 gene confers sensitivity to the tyrosine kinase inhibitor gefitinib. Thus, a diagnostic assay for these mutations will allow for the administration of gefitinib, erlotinib and other tyrosine kinase inhibitors to those patients most likely to respond to the drug.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/294,621, filed Dec. 5, 2005, now U.S. Pat. No. 7,294,468, issued Nov.13, 2007, which is a continuation of International ApplicationPCT/US2005/010645, filed Mar. 31, 2005, which is hereby incorporated byreference in its entirety, which claims benefit under 35 U.S.C. § 119(e)of U.S. Provisional Application Ser. No. 60/558,218 filed Mar. 31, 2004,U.S. Provisional Application Ser. No. 60/561,095 filed Apr. 9, 2004,U.S. Provisional Application Ser. No. 60/565,753 filed Apr. 27, 2004,U.S. Provisional Application No. 60/565,985 filed Apr. 27, 2004, U.S.Provisional Application Ser. No. 60/574,035 filed May 25, 2004, U.S.Provisional Application Ser. No. 60/577,916 filed Jun. 7, 2004 and U.S.Provisional Application Ser. No. 60/592,287 filed Jul. 29, 2004, thecontents of which are herein incorporated by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was supported by the National Institutes for Health (NIH)Grant Nos. RO1 CA 092824, P50 CA 090578, PO1 95281, and 1K12CA87723-01,and the Government of the United States has certain rights thereto.

SEQUENCE LISTING

The instant application contains a “lengthy” Sequence Listing which hasbeen submitted via CD-R in lieu of a printed paper copy, and is herebyincorporated by reference in its entirety. Said CD-R, recorded on Dec.27, 2007, are labeled CRF, “Copy 1” and “Copy 2”, respectively, and eachcontains only one identical 448 KB file (0579562.txt).

BACKGROUND

Epithelial cell cancers, for example, prostate cancer, breast cancer,colon cancer, lung cancer, pancreatic cancer, ovarian cancer, cancer ofthe spleen, testicular cancer, cancer of the thymus, etc., are diseasescharacterized by abnormal, accelerated growth of epithelial cells. Thisaccelerated growth initially causes a tumor to form. Eventually,metastasis to different organ sites can also occur. Although progresshas been made in the diagnosis and treatment of various cancers, thesediseases still result in significant mortality.

Lung cancer remains the leading cause of cancer death in industrializedcountries. Cancers that begin in the lungs are divided into two majortypes, non-small cell lung cancer and small cell lung cancer, dependingon how the cells appear under a microscope. Non-small cell lung cancer(squamous cell carcinoma, adenocarcinoma, and large cell carcinoma)generally spreads to other organs more slowly than does small cell lungcancer. About 75 percent of lung cancer cases are categorized asnon-small cell lung cancer (e.g., adenocarcinomas), and the other 25percent are small cell lung cancer. Non-small cell lung cancer (NSCLC)is the leading cause of cancer deaths in the United States, Japan andWestern Europe. For patients with advanced disease, chemotherapyprovides a modest benefit in survival, but at the cost of significanttoxicity, underscoring the need for therapeutic agents that arespecifically targeted to the critical genetic lesions that direct tumorgrowth (Schiller J H et al., N Engl J Med, 346: 92-98, 2002).

Epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa)membrane-bound protein expressed on the surface of epithelial cells.EGFR is a member of the growth factor receptor family of proteintyrosine kinases, a class of cell cycle regulatory molecules. (W. J.Gullick et al., 1986, Cancer Res., 46:285-292). EGFR is activated whenits ligand (either EGF or TGF-α) binds to the extracellular domain,resulting in autophosphorylation of the receptor's intracellulartyrosine kinase domain (S. Cohen et al., 1980, J. Biol. Chem.,255:483-44842; A. B. Schreiber et al., 1983, J. Biol. Chem.,258:846-853).

EGFR is the protein product of a growth promoting oncogene, erbB orErbB1, that is but one member of a family, i.e., the ERBB family ofprotooncogenes, believed to play pivotal roles in the development andprogression of many human cancers. In particular, increased expressionof EGFR has been observed in breast, bladder, lung, head, neck andstomach cancer as well as glioblastomas. The ERBB family of oncogenesencodes four, structurally-related transmembrane receptors, namely,EGFR, HER-2/neu (erbB2), HER-3 (erbB3) and HER-4 (erbB4). Clinically,ERBB oncogene amplification and/or receptor overexpression in tumorshave been reported to correlate with disease recurrence and poor patientprognosis, as well as with responsiveness in therapy. (L. Harris et al.,1999, Int. J. Biol. Markers, 14:8-15; and J. Mendelsohn and J. Baselga,2000, Oncogene, 19:6550-6565).

EGFR is composed of three principal domains, namely, the extracellulardomain (ECD), which is glycosylated and contains the ligand-bindingpocket with two cysteine-rich regions; a short transmembrane domain, andan intracellular domain that has intrinsic tyrosine kinase activity. Thetransmembrane region joins the ligand-binding domain to theintracellular domain. Amino acid and DNA sequence analysis, as well asstudies of nonglycosylated forms of EGFR, indicate that the proteinbackbone of EGFR has a mass of 132 kDa, with 1186 amino acid residues(A. L. Ullrich et al., 1984, Nature, 307:418-425; J. Downward et al.,1984, Nature, 307:521-527; C. R. Carlin et al., 1986, Mol. Cell. Biol.,6:257-264; and F. L. V. Mayes and M. D. Waterfield, 1984, The EMBO J.,3:531-537).

The binding of EGF or TGF-α to EGFR activates a signal transductionpathway and results in cell proliferation. The dimerization,conformational changes and internalization of EGFR molecules function totransmit intracellular signals leading to cell growth regulation (G.Carpenter and S. Cohen, 1979, Ann. Rev. Biochem., 48:193-216). Geneticalterations that affect the regulation of growth factor receptorfunction, or lead to overexpression of receptor and/or ligand, result incell proliferation. In addition, EGFR has been determined to play a rolein cell differentiation, enhancement of cell motility, proteinsecretion, neovascularization, invasion, metastasis and resistance ofcancer cells to chemotherapeutic agents and radiation. (M.-J. Oh et al.,2000, Clin. Cancer Res., 6:4760-4763).

A variety of inhibitors of EGFR have been identified, including a numberalready undergoing clinical trials for treatment of various cancers. Fora recent summary, see de Bono, J. S. and Rowinsky, E. K. (2002), “TheErbB Receptor Family: A Therapeutic Target For Cancer”, Trends inMolecular Medicine, 8, S19-26.

A promising set of targets for therapeutic intervention in the treatmentof cancer includes the members of the HER-kinase axis. They arefrequently upregulated in solid epithelial tumors of, by way of example,the prostate, lung and breast, and are also upregulated in glioblastomatumors. Epidermal growth factor receptor (EGFR) is a member of theHER-kinase axis, and has been the target of choice for the developmentof several different cancer therapies. EGFR tyrosine kinase inhibitors(EGFR-TKIs) are among these therapies, since the reversiblephosphorylation of tyrosine residues is required for activation of theEGFR pathway. In other words, EGFR-TKIs block a cell surface receptorresponsible for triggering and/or maintaining the cell signaling pathwaythat induces tumor cell growth and division. Specifically, it isbelieved that these inhibitors interfere with the EGFR kinase domain,referred to as HER-1. Among the more promising EGFR-TKIs are threeseries of compounds: quinazolines, pyridopyrimidines andpyrrolopyrimidines.

Two of the more advanced compounds in clinical development includeGefitinib (compound ZD1839 developed by AstraZeneca UK Ltd.; availableunder the tradename IRESSA; hereinafter “IRESSA”) and Erlotinib(compound OSI-774 developed by Genentech, Inc. and OSI Pharmaceuticals,Inc.; available under the tradename TARCEVA; hereinafter “TARCEVA”);both have generated encouraging clinical results. Conventional cancertreatment with both IRESSA and TARCEVA involves the daily, oraladministration of no more than 500 mg of the respective compounds. InMay, 2003, IRESSA became the first of these products to reach the UnitedStates market, when it was approved for the treatment of advancednon-small cell lung cancer patients.

IRESSA is an orally active quinazoline that functions by directlyinhibiting tyrosine kinase phosphorylation on the EGFR molecule. Itcompetes for the adenosine triphosphate (ATP) binding site, leading tosuppression of the HER-kinase axis. The exact mechanism of the IRESSAresponse is not completely understood, however, studies suggest that thepresence of EGFR is a necessary prerequisite for its action.

A significant limitation in using these compounds is that recipientsthereof may develop a resistance to their therapeutic effects after theyinitially respond to therapy, or they may not respond to EGFR-TKIs toany measurable degree at all. In fact, only 10-15 percent of advancednon-small cell lung cancer patients respond to EGFR kinase inhibitors.Thus, a better understanding of the molecular mechanisms underlyingsensitivity to IRESSA and TARCEVA would be extremely beneficial intargeting therapy to those individuals whom are most likely to benefitfrom such therapy.

There is a significant need in the art for a satisfactory treatment ofcancer, and specifically epithelial cell cancers such as lung, ovarian,breast, brain, colon and prostate cancers, which incorporates thebenefits of TKI therapy and overcoming the non-responsiveness exhibitedby patients. Such a treatment could have a dramatic impact on the healthof individuals, and especially older individuals, among whom cancer isespecially common.

SUMMARY

Tyrosine kinase inhibitor (TKI) therapy such as gefitinib (IRESSA®) isnot effective in the vast majority of individuals that are affected withthe cancers noted above. The present inventors have surprisinglydiscovered that the presence of somatic mutations in the kinase domainof EGFR substantially increases sensitivity of the EGFR to TKI such asIRESSA, TARCEVA. For example less than 30% of patients having suchcancer are susceptible to treatment by current TKIs, whereas greaterthan 50%, more preferably 60, 70, 80, 90% of patients having a mutationin the EGFR kinase domain are susceptible. In addition, these mutationsconfer increased kinase activity of the EGFR. Thus, patients havingthese mutations will likely be responsive to current tyrosine kinaseinhibitor (TKI) therapy, for example, gefitinib.

Accordingly, the present invention provides a novel method to determinethe likelihood of effectiveness of an epidermal growth factor receptor(EGFR) targeting treatment in a human patient affected with cancer. Themethod comprises detecting the presence or absence of at least onenucleic acid variance in the kinase domain of the erbB1 gene of saidpatient relative to the wildtype erbB1 gene. The presence of at leastone variance indicates that the EGFR targeting treatment is likely to beeffective. Preferably, the nucleic acid variance increases the kinaseactivity of the EGFR. The patient can then be treated with an EGFRtargeting treatment. In one embodiment of the present invention, theEGFR targeting treatment is a tyrosine kinase inhibitor. In a preferredembodiment, the tyrosine kinase inhibitor is an anilinoquinazoline. Theanilinoquinazoline may be a synthetic anilinoquinazoline. Preferably,the synthetic anilinoquinazoline is either gefitinib or erlotinib. Inanother embodiment, the EGFR targeting treatment is an irreversible EGFRinhibitor, including 4-dimethylamino-but-2-enoic acid[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide(“EKB-569”, sometimes also referred to as “EKI-569”, see for exampleWO/2005/018677 and Torrance et al., Nature Medicine, vol. 6, No. 9,September 2000, p. 1024) and/or HKI-272 or HKI-357 (Wyeth; seeGreenberger et al., Proc. 11^(th) NCI EORTC-AACR Symposium on New Drugsin Cancer Therapy, Clinical Cancer Res. Vol. 6 Supplement, November2000, ISSN 1078-0432; in Rabindran et al., Cancer Res. 64: 3958-3965(2004); Holbro and Hynes, Ann. Rev. Pharm. Tox. 44:195-217 (2004); Tsouet al, j. Med. Chem. 2005, 48, 1107-1131; and Tejpar et al., J. Clin.Oncol. ASCO Annual Meeting Proc. Vol. 22, No. 14S: 3579 (2004)).

In one embodiment of the present invention, the EGFR is obtained from abiological sample from a patient with or at risk for developing cancer.The variance in the kinase domain of EGFR (or the erbB1 gene) effectsthe conformational structure of the ATP-binding pocket. Preferably, thevariance in the kinase domain of EGFR is an in frame deletion or asubstitution in exon 18, 19, 20 or 21.

In one embodiment, the in frame deletion is in exon 19 of EGFR (erbB1).The in frame deletion in exon 19 preferably comprises at deletion of atleast amino acids leucine, arginine, glutamic acid and alanine, atcodons 747, 748, 749, and 750. In one embodiment, the in-frame deletioncomprises nucleotides 2481 to 2495 of SEQ ID NO: 511, and deletes aminoacids 746 to 750 (the sequence glutamic acid, leucine, arginine,glutamic acid, and alanine), see Table 2, Table S2, FIG. 2B, FIG. 4A,FIG. 5, SEQ ID NO: 511, FIG. 6C, and FIG. 8C. In another embodiment, thein-frame deletion comprises nucleotides 2482 to 2496 of SEQ ID NO: 511,and deletes amino acids 746 to 750, see Table S2, FIG. 5, SEQ ID NO:511, and FIG. 6C. Alternatively, the in-frame deletion comprisesnucleotides 2486 to 2497 of SEQ ID NO: 511, see Table 2, FIG. 2C, FIG.4A, FIG. 5, SEQ ID NO: 511, or nucleotides 2486 to 2503 of SEQ ID NO:511, see Table 2, Table S3A, FIG. 2A, FIG. 4A, FIG. 5, SEQ ID NO: 511,FIG. 6C, and FIG. 8E. Alternatively, the in-frame deletion comprisesnucleotides 2485 to 2493 of SEQ ID NO: 511, together with a substitutionof cytosine for guanine at nucleotide 2494 of SEQ ID NO: 511, see TableS3A and FIG. 8D, or a deletion of nucleotides 2489-2501 OF SEQ ID NO:511, together with a substitution of thymine for adenine at nucleotide2483 of SEQ ID NO: 511, see Table S3A and FIG. 8F, or a deletion ofnucleotides 2500 to 2523 of SEQ ID NO: 511, see Table S2 (SEQ ID NO:437). Alternatively, the in-frame deletion comprises nucleotides 2485 to2496 of SEQ ID NO: 511, delTTAAGAGAAGCA (SEQ ID NO: 554); 2251A>C, or2240-2250delTAAGAGAAGCA (SEQ ID NO: 720), or 2257-2271delCCGAAAGCCAACAAG(SEQ ID NO: 721), as shown in Table S3B.

In another embodiment, the substitution is in exon 21 of EGFR. Thesubstitution in exon 21 comprises at least one amino acid. In oneembodiment, the substitution in exon 21 comprises a substitution of aguanine for a thymine at nucleotide 2818 of SEQ ID NO: 511, see FIG. 4Aand FIG. 5, SEQ ID NO: 511. This substitution results in an amino acidsubstitution, where the wildtype Leucine is replaced with an Arginine atamino acid 858, see FIG. 5, Table 2, Table S2, Table S3A, FIG. 2D, FIG.6A, FIG. 8B, and SEQ ID NO: 512. Alternatively, the substitution in exon21 comprises a substitution of an adenine for a thymine at nucleotide2827 of SEQ ID NO: 511, see FIG. 4A and FIG. 5, SEQ ID NO: 511. Thissubstitution results in an amino acid substitution, where the wildtypeLeucine is replaced with a Glutamine at amino acid 861, see FIG. 5 (SEQID NOS 740-762, respectively, in order of appearance), Table 2 (SEQ IDNOS 730-739, respectively, in order of appearance), FIG. 2E, Table S3B(SEQ ID NOS 554 & 720-729, respectively, in order of appearance), andSEQ ID NO: 512.

The substitution may also be in exon 18 of EGFR. In one embodiment, thesubstitution is in exon 18 is a thymine for a guanine at nucleotide 2400of SEQ ID NO: 511, see FIG. 4A and FIG. 5, SEQ ID NO: 511. Thissubstitution results in an amino acid substitution, where the wildtypeGlycine is substituted with a Cysteine at codon 719, see FIG. 5, SEQ IDNO: 512. In another embodiment, the substitution in exon 18 is anadenine for a guanine at nucleotide 2400 of SEQ ID NO: 511 resulting inan amino acid substitution, where the wildtype Glycine is substitutedfor a Serine at codon 719, see Table S2, FIG. 6B, FIG. 8A, FIG. 5, SEQID NO: 511 and 512.

In another embodiment, the substitution is an insertion of guanine,guanine and thymine (GGT) after nucleotide 2561 and before nucleotide2562 of SEQ ID NO: 511 (2561-2562 ins GGT). This can also be describedas an insertion of valine (V) at amino acid 772 (P772_H733 insV). Othermutations are shown in Table S3B and include, for example, and insertionof CAACCCGG after nucleotide 2554 and before nucleotide 2555 of SEQ IDNO 511 and an insertion of GCGTGGACA after nucleotide 2556 and beforenucleotide 2557 of SEQ ID NO 511. The substitution may also be in exon20 and in one embodiment is a substitution of AA for GG at nucleotides2579 and 2580 of SEQ ID NO: 511, see Table S3B.

In summary, in preferred embodiments, the nucleic acid variance of theerbB1 gene is a substitution of a thymine for a guanine or an adeninefor a guanine at nucleotide 2400 of SEQ ID NO 511, a deletion ofnucleotides 2481-2495, 2486-2497, 2486-2503, 2482-2496, 2500-2523, or2482-2490 of SEQ ID NO 511, an insertion of nucleotides guanine,guanine, and thymine (GGT) after nucleotide 2561 and before nucleotide2562 of SEQ ID NO 511, and a substitution of a guanine for a thymine atnucleotide 2818 or an adenine for a thymine at nucleotide 2828 of SEQ IDNO 511.

The detection of the presence or absence of at least one nucleic acidvariance can be determined by amplifying a segment of nucleic acidencoding the receptor. The segment to be amplified is 1000 nucleotidesin length, preferably, 500 nucleotides in length, and most preferably100 nucleotides in length or less. The segment to be amplified caninclude a plurality of variances.

In another embodiment, the detection of the presence or absence of atleast one variance provides for contacting EGFR nucleic acid containinga variance site with at least one nucleic acid probe. The probepreferentially hybridizes with a nucleic acid sequence including avariance site and containing complementary nucleotide bases at thevariance site under selective hybridization conditions. Hybridizationcan be detected with a detectable label.

In yet another embodiment, the detection of the presence or absence ofat least one variance comprises sequencing at least one nucleic acidsequence and comparing the obtained sequence with the known erbB1nucleic acid sequence. Alternatively, the presence or absence of atleast one variance comprises mass spectrometric determination of atleast one nucleic acid sequence.

In a preferred embodiment, the detection of the presence or absence ofat least one nucleic acid variance comprises performing a polymerasechain reaction (PCR). The erbB1 nucleic acid sequence containing thehypothetical variance is amplified and the nucleotide sequence of theamplified nucleic acid is determined. Determining the nucleotidesequence of the amplified nucleic acid comprises sequencing at least onenucleic acid segment. Alternatively, amplification products can analyzedby using any method capable of separating the amplification productsaccording to their size, including automated and manual gelelectrophoresis and the like.

Alternatively, the detection of the presence or absence of at least onevariance comprises determining the haplotype of a plurality of variancesin a gene.

In another embodiment, the presence or absence of an EGFR variance canbe detected by analyzing the erbB1 gene product (protein). In thisembodiment, a probe that specifically binds to a variant EGFR isutilized. In a preferred embodiment, the probe is an antibody thatpreferentially binds to a variant EGFR. The presence of a variant EGFRpredicts the likelihood of effectiveness of an EGFR targeting treatment.Alternatively, the probe may be an antibody fragment, chimeric antibody,humanized antibody or an aptamer.

The present invention further provides a probe which specifically bindsunder selective binding conditions to a nucleic acid sequence comprisingat least one nucleic acid variance in the EGFR gene (erbB1). In oneembodiment, the variance is a mutation in the kinase domain of erbB1that confers a structural change in the ATP-binding pocket.

The probe of the present invention may comprise a nucleic acid sequenceof about 500 nucleotide bases, preferably about 100 nucleotides bases,and most preferably about 50 or about 25 nucleotide bases or fewer inlength. The probe may be composed of DNA, RNA, or peptide nucleic acid(PNA). Furthermore, the probe may contain a detectable label, such as,for example, a fluorescent or enzymatic label.

The present invention additionally provides a novel method to determinethe likelihood of effectiveness of an epidermal growth factor receptor(EGFR) targeting treatment in a patient affected with cancer. The methodcomprises determining the kinase activity of the EGFR in a biologicalsample from a patient. An increase in kinase activity followingstimulation with an EGFR ligand, compared to a normal control, indicatesthat the EGFR targeting treatment is likely to be effective.

The present invention further provides a novel method for treating apatient affected with or at risk for developing cancer. The methodinvolves determining whether the kinase domain of the EGFR of a patientcontains at least one nucleic acid variance. Preferably, the EGFR islocated at the site of the tumor or cancer and the nucleic acid varianceis somatic. The presence of such a variance indicates that an EGFRtargeted treatment will be effective. If the variance is present, thetyrosine kinase inhibitor is administered to the patient.

As above, the tyrosine kinase inhibitor administered to an identifiedpatient may be an anilinoquinazoline or an irreversible tyrosine kinaseinhibitor, such as for example, EKB-569, HKI-272 and/or HKI-357 (Wyeth).Preferably, the anilinoquinazoline is a synthetic anilinoquinazoline andmost preferably the synthetic anilinoquinazoline is gefitinib anderlotinib.

The cancer to be treated by the methods of the present inventioninclude, for example, but are not limited to, gastrointestinal cancer,prostate cancer, ovarian cancer, breast cancer, head and neck cancer,lung cancer, non-small cell lung cancer, cancer of the nervous system,kidney cancer, retina cancer, skin cancer, liver cancer, pancreaticcancer, genital-urinary cancer and bladder cancer. In a preferredembodiment, the cancer is non-small cell lung cancer.

A kit for implementing the PCR methods of the present invention is alsoencompassed. The kit includes at least one degenerate primer pairdesigned to anneal to nucleic acid regions bordering the genes thatencode for the ATP-binding pocket of the EGFR kinase domain.Additionally, the kit contains the products and reagents required tocarry out PCR amplification, and instructions.

In a preferred embodiment, the primer pairs contained within the kit areselected from the group consisting of SEQ ID NO: 505, SEQ ID NO: 506,SEQ ID NO: 507, and SEQ ID NO: 508. Also preferred are the primerslisted in Table 6 and 7 in the examples.

In yet another embodiment, the present invention discloses a method forselecting a compound that inhibits the catalytic kinase activity of avariant epidermal growth factor receptor (EGFR). As a first step, avariant EGFR is contacted with a potential compound. The resultantkinase activity of the variant EGFR is then detected and a compound isselected that inhibits the kinase activity of the variant EGFR. In oneembodiment, the variant EGFR is contained within a cell. The method canalso be used to select a compound that inhibits the kinase activity of avariant EGFR having a secondary mutation in the kinase domain thatconfers resistance to a TKI, e.g., gefitinib or erlotinib.

In one embodiment, the variant EGFR is labeled. In another embodiment,the EGFR is bound to a solid support. In a preferred embodiment, thesolid support is a protein chip.

In yet another embodiment of the present invention, a pharmaceuticalcomposition that inhibits the catalytic kinase activity of a variantepidermal growth factor receptor (EGFR) is disclosed. The compound thatinhibits the catalytic kinase activity of a variant EGFR is selectedfrom the group consisting of an antibody, antibody fragment, smallmolecule, peptide, protein, antisense nucleic acid, ribozyme, PNA,siRNA, oligonucleotide aptamer, and peptide aptamer.

A method for treating a patient having an EGFR mediated disease is alsodisclosed. In accordance with the method, the patient is administeredthe pharmaceutical composition that inhibits the catalytic kinaseactivity of a variant epidermal growth factor receptor (EGFR).

In one embodiment, the EGFR mediated disease is cancer. In a preferredembodiment, the cancer is of epithelial origin. For example, the canceris gastrointestinal cancer, prostate cancer, ovarian cancer, breastcancer, head and neck cancer, lung cancer, non-small cell lung cancer,cancer of the nervous system, kidney cancer, retina cancer, skin cancer,liver cancer, pancreatic cancer, genital-urinary cancer and bladdercancer. In a preferred embodiment, the cancer is non-small cell lungcancer.

In another embodiment, a method for predicting the acquisition ofsecondary mutations (or selecting for mutations) in the kinase domain ofthe erbB1 gene is disclosed. A cell expressing a variant form of theerbB1 gene is contacted with an effective, yet sub-lethal dose of atyrosine kinase inhibitor. Cells that are resistant to a growth arresteffect of the tyrosine kinase inhibitor are selected and the erbB1nucleic acid is analyzed for the presence of additional mutations in theerbB1 kinase domain. In one embodiment, the cell is in vitro. In anotherembodiment, the cell is obtained from a transgenic animal. In oneembodiment, the transgenic animal is a mouse. In this mouse model, cellsto be studied are obtained from a tumor biopsy. Cells containing asecondary mutation in the erbB1 kinase domain selected by the presentinvention can be used in the above methods to select a compound thatinhibits the kinase activity of the variant EGFR having a secondarymutation in the kinase domain.

In an alternative embodiment for predicting the acquisition of secondarymutations in the kinase domain of the erbB1 gene, cells expressing avariant form of the erbB1 gene are first contacted with an effectiveamount of a mutagenizing agent. The mutagenizing is, for example, ethylmethanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU),N-methyl-N-nitrosourea (MNU), phocarbaxine hydrochloride (Prc), methylmethanesulfonate (MeMS), chlorambucil (Chl), melphalan, porcarbazinehydrochloride, cyclophosphamide (Cp), diethyl sulfate (Et₂SO₄),acrylamide monomer (AA), triethylene melamin (TEM), nitrogen mustard,vincristine, dimethylnitrosamine, N-methyl-N′-nitro-Nitrosoguanidine(MNNG), 7,12 dimethylbenz(a)anthracene (DMBA), ethylene oxide,hexamethylphosphoramide, bisulfan, or ethyl methanesulforate (EtMs). Thecell is then contacted with an effective, yet sub-lethal dose of atyrosine kinase inhibitor. Cells that are resistant to a growth arresteffect of the tyrosine kinase inhibitor are selected and the erbB1nucleic acid is analyzed for the presence of additional mutations in theerbB1 kinase domain.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show a representative illustration of Gefitinib response inrefractory non-small cell lung cancer (NSCLC). Chest CT scan of case 6(Table 1), demonstrating (FIG. 1A) a large mass in the right lung beforetreatment with gefitinib, and (FIG. 1B) marked improvement six weeksafter Gefitinib was initiated.

FIGS. 2A-2F show EGFR mutations in Gefitinib-responsive tumors.

FIGS. 2A-2C show nucleotide sequence of the EGFR gene in tumor specimenswith heterozygous in-frame deletions within the kinase domain (doublepeaks) (SEQ ID NOS 643, 644 and 690-699, respectively, in order ofappearance). Tracings in both sense and antisense directions are shownto demonstrate the two breakpoints of the deletion; wild-type nucleotidesequence is shown in capital letters, and the mutant sequence is inlowercase letters. The 5′ breakpoint of the delL747-T751 insS mutationis preceded by a T to C substitution that does not alter the encodedamino acid.

FIG. 2D and FIG. 2E show heterozygous missense mutations (arrows)resulting in amino acid substitutions within the tyrosine kinase domain(SEQ ID NOS 701 & 703). The double peaks represent two nucleotides atthe site of heterozygous mutations. For comparison, the correspondingwild-type sequence is also shown (SEQ ID NOS 700 & 702).

FIG. 2F is a schematic representation of dimerized EGFR molecules boundby the EGF ligand. The extracellular domain (containing two receptorligand [L]-domains and a furin-like domain), transmembrane region, andthe cytoplasmic domain (containing the catalytic kinase domain) arehighlighted. The position of tyrosine¹⁰⁶⁸ (−1068), a site ofautophosphorylation used as a marker of receptor activation, isindicated, along with downstream effectors activated by EGFRautophosphorylation (STAT3, MAP Kinase (MAPK), and AKT). The location oftumor-associated mutations, all within the tyrosine kinase domain, isshown.

FIGS. 3A-3D demonstrate enhanced EGF-dependent activation of mutant EGFRand increased sensitivity of mutant EGFR to Gefitinib.

FIG. 3A shows a time course of ligand-induced activation of thedelL747-P753insS and L858R mutants, compared with wild type EGFR,following addition of EGF to serum starved cells. EGFRautophosphorylation is used as a marker of receptor activation, usingWestern blotting with an antibody that specifically recognizes thephosphorylated tyrosine¹⁰⁶⁸ residue of EGFR (left panel), compared withthe total levels of EGFR expressed in Cos-7 cells (control; rightpanel). Autophosphorylation of EGFR is measured at intervals followingaddition of EGF (10 ng/ml).

FIG. 3B is a graphical representation of EGF-induction of wild-type andmutant receptor phosphorylation (see panel A). Autoradiographs fromthree independent experiments were quantified using the NIH imagesoftware; intensity of EGFR phosphorylation is normalized to totalprotein expression, and shown as percent activation of the receptor,with standard deviation.

FIG. 3C shows a dose-dependent inhibition of EGFR activation byGefitinib. Autophosphorylation of EGFR tyrosine¹⁰⁶⁸ is demonstrated byWestern blotting analysis of Cos-7 cells expressing wild-type or mutantreceptors, and stimulated with 100 ng/ml of EGF for 30 min. Cells wereuntreated (U) or pretreated for 3 hrs with increasing concentrations ofGefitinib as shown (left panel). Total amounts of EGFR protein expressedare shown as control (right panel).

FIG. 3D shows the quantification of results from two experimentsdescribed for panel 3C (NIH image software). Concentrations ofphosphorylated EGFR were normalized to protein expression levels andexpressed as percent activation of the receptor.

FIGS. 4A-4C demonstrate clustering of mutations at critical sites withinthe ATP-binding pocket of EGFR.

FIG. 4A shows the position of overlapping in-frame deletions in exon 19and missense mutations in exon 21 of the EGFR gene, in multiple cases ofNSCLC (SEQ ID NOS 495-504 (DNA)). Partial nucleotide sequence is shownfor each exon, with deletions marked by dashed lines and missensemutations highlighted and underlined; the wild-type EGFR nucleotide andamino acid sequences are shown (SEQ ID NOS 493 & 494 (DNA) & 509-510(amino acid)).

FIG. 4B shows the tridimensional structure of the EGFR ATP cleft flankedby the amino (N) and carboxy (C) lobes of the kinase domain (coordinatesderived from PDB 1M14, and displayed using Cn3D software). Theinhibitor, representing Gefitinib, is pictured occupying the ATP cleft.The locations of the two missense mutations are shown, within theactivating loop of the kinase; the three in-frame deletions are allpresent within another loop, which flanks the ATP cleft.

FIG. 4C is a close-up of the EGFR kinase domain, showing the criticalamino acid residues implicated in binding to either ATP or to theinhibitor. Specifically, 4-anilinoquinazoline compounds such asgefitinib inhibit catalysis by occupying the ATP-binding site, wherethey form hydrogen bonds with methionine⁷⁹³ (M793) and cysteine⁷⁷⁵(C775) residues, whereas their anilino ring is close to methionine⁷⁶⁶(M766), lysine⁷⁴⁵ (K745), and leucine⁷⁸⁸ (L788) residues. In-framedeletions within the loop that is targeted by mutations are predicted toalter the position of these amino acids relative to the inhibitor.Mutated residues are shown within the activation loop of the tyrosinekinase.

FIG. 5 shows the nucleotide and amino acid sequence of the erbB1 gene.The amino acids are depicted as single letters, known to those of skillin the art. Nucleotide variances in the kinase domain are highlighted bypatient number, see Table 2. SEQ ID NO: 511 includes nucleotides 1through 3633. SEQ ID NO: 512 includes amino acids 1 through 1210.

FIGS. 6A-6C: Sequence alignment of selected regions within the EGFR andB-Raf kinase domains. Depiction of EGFR mutations in human NSCLC. EGFR(gb:X00588;) mutations in NSCLC tumors are highlighted in gray. B-Raf(gb:M95712) mutations in multiple tumor types (5) are highlighted inblack. Asterisks denote residues conserved between EGFR and B-Raf. FIG.6A depicts L858R mutations in the activation loop (SEQ ID NOS 477-479).FIG. 6B depicts the G719S mutant in the P-loop (SEQ ID NOS 480-482).FIG. 6C depicts deletion mutants in EGFR exon 19 (SEQ ID NOS 483-489).

FIG. 7: Positions of missense mutations G719S and L858R and the Del-1deletion in the three-dimensional structure of the EGFR kinase domain.The activation loop is shown in yellow, the P-loop is in blue and theC-lobe and N-lobe are as indicated. The residues targeted by mutation ordeletion are highlighted in red. The Del-1 mutation targets the residuesELREA in codons 746 to 750. The mutations are located in highlyconserved regions within kinases and are found in the p-loop andactivation loop, which surround the region where ATP and also gefitiniband erlotinib are predicted to bind.

FIGS. 8A-8F. Representative chromatograms of EGFR DNA from normal tissueand from tumor tissues. The locations of the identified mutations are asfollows. FIG. 8A depicts the Exon 18 Kinase domain P loop (SEQ ID NOS704-705). Arrow depicts mutation in nucleic acid sequence G2400A(corresponding to SEQ ID NO: 511) that results in amino acid changeG719S FIG. 8B depicts the Exon 21 Kinase domain A-loop (SEQ ID NOS706-707). Arrow depicts mutation in nucleic acid sequence T2573G(corresponding to SEQ ID NO: 511) that results in amino acid changeL858R. FIG. 8C depicts the Exon 19 Kinase domain Del-1 (SEQ ID NOS708-710). FIG. 8D depicts the Exon 19 Kinase domain Del-3 (SEQ ID NOS711-713). FIG. 8E depicts the Exon 19 Kinase domain Del-4 (SEQ ID NOS714-716). FIG. 8F depicts the Exon 19 Kinase domain Del-5 (SEQ ID NOS717-719).

FIG. 9: Sequence alignment of the EGFR and BCR-ABL polypeptides and thelocation of residues conferring a drug resistant phenotype. The EGFRpolypeptide (SEQ ID NO:492) encoded by the nucleotide sequence disclosedin GenBank accno. NM_(—)005228 and the BCR-ABL polypeptide (SEQ IDNO:491) encoded by the nucleotide sequence disclosed in GenBank accno.M14752 are aligned and conserved residues are shaded. BCR-ABL mutationsconferring resistance to the tyrosine kinase inhibitor imatinib (STI571,Glivec/Gleevec) are denoted by asterisks.

FIG. 10 shows the decision making process for patient with metastaticNSCLC undergoing EGFR testing.

FIG. 11 shows a diagram of EGFR exons 18-24 (not to scale). Arrowsdepict the location of identified mutations. Astericks denote the numberof patients with mutations at each location. The blow-up diagram depictsthe overlap of the exon 19 deletions, and the number of patients (n)with each deletion (nucleotides 2479-2523 of SEQ ID NO: 511 and residues745-759 of SEQ ID NO: 512). Note that these are the results are notmeant to be inclusive of all the EGFR mutations to date.

DETAILED DESCRIPTION

The present invention provides a novel method to determine thelikelihood of effectiveness of an epidermal growth factor receptor(EGFR) targeting treatment in a patient affected with cancer. The methodcomprises detecting the presence or absence of at least one nucleic acidvariance in the kinase domain of the erbB1 gene of said patient. Thepresence of at least one variance indicates that the EGFR targetingtreatment is likely to be effective. Preferably, the nucleic acidvariance increases the kinase activity of the EGFR. The patient can thenbe treated with an EGFR targeting treatment. In one embodiment of thepresent invention, the EGFR targeting treatment is a tyrosine kinaseinhibitor. In a preferred embodiment, the tyrosine kinase inhibitor isan anilinoquinazoline. The anilinoquinazoline may be a syntheticanilinoquinazoline. Preferably, the synthetic anilinoquinazoline iseither gefitinib or erlotinib.

Definitions

The terms “ErbB1”, “epidermal growth factor receptor” and “EGFR” areused interchangeably herein and refer to native sequence EGFR asdisclosed, for example, in Carpenter et al. Ann. Rev. Biochem.56:881-914 (1987), including variants thereof (e.g. a deletion mutantEGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 refersto the gene encoding the EGFR protein product.

The term “kinase activity increasing nucleic acid variance” as usedherein refers to a variance (i.e. mutation) in the nucleotide sequenceof a gene that results in an increased kinase activity. The increasedkinase activity is a direct result of the variance in the nucleic acidand is associated with the protein for which the gene encodes.

The term “drug” or “compound” as used herein refers to a chemical entityor biological product, or combination of chemical entities or biologicalproducts, administered to a person to treat or prevent or control adisease or condition. The chemical entity or biological product ispreferably, but not necessarily a low molecular weight compound, but mayalso be a larger compound, for example, an oligomer of nucleic acids,amino acids, or carbohydrates including without limitation proteins,oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs,lipoproteins, aptamers, and modifications and combinations thereof.

The term “genotype” in the context of this invention refers to theparticular allelic form of a gene, which can be defined by theparticular nucleotide(s) present in a nucleic acid sequence at aparticular site(s).

The terms “variant form of a gene”, “form of a gene”, or “allele” referto one specific form of a gene in a population, the specific formdiffering from other forms of the same gene in the sequence of at leastone, and frequently more than one, variant sites within the sequence ofthe gene. The sequences at these variant sites that differ betweendifferent alleles of the gene are termed “gene sequence variances” or“variances” or “variants”. Other terms known in the art to be equivalentinclude mutation and polymorphism, although mutation is often used torefer to an allele associated with a deleterious phenotype. In preferredaspects of this invention, the variances are selected from the groupconsisting of the variances listed in the variance tables herein.

In the context of this invention, the term “probe” refers to a moleculewhich can detectably distinguish between target molecules differing instructure. Detection can be accomplished in a variety of different waysdepending on the type of probe used and the type of target molecule.Thus, for example, detection may be based on discrimination of activitylevels of the target molecule, but preferably is based on detection ofspecific binding. Examples of such specific binding include antibodybinding and nucleic acid probe hybridization. Thus, for example, probescan include enzyme substrates, antibodies and antibody fragments, andpreferably nucleic acid hybridization probes.

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. “Lesseffective” means that the treatment results in a therapeuticallysignificant lower level of pharmacological effectiveness and/or atherapeutically greater level of adverse physiological effects.

The term “primer”, as used herein, refers to an oligonucleotide which iscapable of acting as a point of initiation of polynucleotide synthesisalong a complementary strand when placed under conditions in whichsynthesis of a primer extension product which is complementary to apolynucleotide is catalyzed. Such conditions include the presence offour different nucleotide triphosphates or nucleoside analogs and one ormore agents for polymerization such as DNA polymerase and/or reversetranscriptase, in an appropriate buffer (“buffer” includes substituentswhich are cofactors, or which affect pH, ionic strength, etc.), and at asuitable temperature. A primer must be sufficiently long to prime thesynthesis of extension products in the presence of an agent forpolymerase. A typical primer contains at least about 5 nucleotides inlength of a sequence substantially complementary to the target sequence,but somewhat longer primers are preferred. Usually primers contain about15-26 nucleotides, but longer primers may also be employed.

A primer will always contain a sequence substantially complementary tothe target sequence, that is the specific sequence to be amplified, towhich it can anneal. A primer may, optionally, also comprise a promotersequence. The term “promoter sequence” defines a single strand of anucleic acid sequence that is specifically recognized by an RNApolymerase that binds to a recognized sequence and initiates the processof transcription by which an RNA transcript is produced. In principle,any promoter sequence may be employed for which there is a known andavailable polymerase that is capable of recognizing the initiationsequence. Known and useful promoters are those that are recognized bycertain bacteriophage polymerases, such as bacteriophage T3, T7 or SP6.

A “microarray” is a linear or two-dimensional array of preferablydiscrete regions, each having a defined area, formed on the surface of asolid support. The density of the discrete regions on a microarray isdetermined by the total numbers of target polynucleotides to be detectedon the surface of a single solid phase support, preferably at leastabout 50/cm², more preferably at least about 100/cm², even morepreferably at least about 500/cm², and still more preferably at leastabout 1,000/cm². As used herein, a DNA microarray is an array ofoligonucleotide primers placed on a chip or other surfaces used toamplify or clone target polynucleotides. Since the position of eachparticular group of primers in the array is known, the identities of thetarget polynucleotides can be determined based on their binding to aparticular position in the microarray.

The term “label” refers to a composition capable of producing adetectable signal indicative of the presence of the targetpolynucleotide in an assay sample. Suitable labels includeradioisotopes, nucleotide chromophores, enzymes, substrates, fluorescentmolecules, chemiluminescent moieties, magnetic particles, bioluminescentmoieties, and the like. As such, a label is any composition detectableby spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means.

The term “support” refers to conventional supports such as beads,particles, dipsticks, fibers, filters, membranes and silane or silicatesupports such as glass slides.

The term “amplify” is used in the broad sense to mean creating anamplification product which may include, for example, additional targetmolecules, or target-like molecules or molecules complementary to thetarget molecule, which molecules are created by virtue of the presenceof the target molecule in the sample. In the situation where the targetis a nucleic acid, an amplification product can be made enzymaticallywith DNA or RNA polymerases or reverse transcriptases.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from an individual, including but not limited to, forexample, blood, plasma, serum, tumor biopsy, urine, stool, sputum,spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the externalsections of the skin, respiratory, intestinal, and genitourinary tracts,tears, saliva, milk, cells (including but not limited to blood cells),tumors, organs, and also samples of in vitro cell culture constituent.In a preferred embodiment, the sample is from a resection, bronchoscopicbiopsy, or core needle biopsy of a primary or metastatic tumor, or acellblock from pleural fluid. In addition, fine needle aspirate samplesare used. Samples may be either paraffin-embedded or frozen tissue.

The term “antibody” is meant to be an immunoglobulin protein that iscapable of binding an antigen. Antibody as used herein is meant toinclude antibody fragments, e.g. F(ab′)2, Fab′, Fab, capable of bindingthe antigen or antigenic fragment of interest. Preferably, the bindingof the antibody to the antigen inhibits the activity of a variant formof EGFR.

The term “humanized antibody” is used herein to describe completeantibody molecules, i.e. composed of two complete light chains and twocomplete heavy chains, as well as antibodies consisting only of antibodyfragments, e.g. Fab, Fab′, F (ab′) 2, and Fv, wherein the CDRs arederived from a non-human source and the remaining portion of the Igmolecule or fragment thereof is derived from a human antibody,preferably produced from a nucleic acid sequence encoding a humanantibody.

The terms “human antibody” and “humanized antibody” are used herein todescribe an antibody of which all portions of the antibody molecule arederived from a nucleic acid sequence encoding a human antibody. Suchhuman antibodies are most desirable for use in antibody therapies, assuch antibodies would elicit little or no immune response in the humanpatient.

The term “chimeric antibody” is used herein to describe an antibodymolecule as well as antibody fragments, as described above in thedefinition of the term “humanized antibody.” The term “chimericantibody” encompasses humanized antibodies. Chimeric antibodies have atleast one portion of a heavy or light chain amino acid sequence derivedfrom a first mammalian species and another portion of the heavy or lightchain amino acid sequence derived from a second, different mammalianspecies.

Preferably, the variable region is derived from a non-human mammalianspecies and the constant region is derived from a human species.Specifically, the chimeric antibody is preferably produced from a 9nucleotide sequence from a non-human mammal encoding a variable regionand a nucleotide sequence from a human encoding a constant region of anantibody.

Table 2 is a partial list of DNA sequence variances in the kinase domainof erbB1 relevant to the methods described in the present invention.These variances were identified by the inventors in studies ofbiological samples from patients with NSCLC who responded to gefitiniband patients with no exposure to gefitinb.

Nucleic acid molecules can be isolated from a particular biologicalsample using any of a number of procedures, which are well-known in theart, the particular isolation procedure chosen being appropriate for theparticular biological sample. For example, freeze-thaw and alkalinelysis procedures can be useful for obtaining nucleic acid molecules fromsolid materials; heat and alkaline lysis procedures can be useful forobtaining nucleic acid molecules from urine; and proteinase K extractioncan be used to obtain nucleic acid from blood (Rolff, A et al. PCR:Clinical Diagnostics and Research, Springer (1994).

Detection Methods

Determining the presence or absence of a particular variance orplurality of variances in the kinase domain of the erbB1 gene in apatient with or at risk for developing cancer can be performed in avariety of ways. Such tests are commonly performed using DNA or RNAcollected from biological samples, e.g., tissue biopsies, urine, stool,sputum, blood, cells, tissue scrapings, breast aspirates or othercellular materials, and can be performed by a variety of methodsincluding, but not limited to, PCR, hybridization with allele-specificprobes, enzymatic mutation detection, chemical cleavage of mismatches,mass spectrometry or DNA sequencing, including minisequencing. Inparticular embodiments, hybridization with allele specific probes can beconducted in two formats: (1) allele specific oligonucleotides bound toa solid phase (glass, silicon, nylon membranes) and the labeled samplein solution, as in many DNA chip applications, or (2) bound sample(often cloned DNA or PCR amplified DNA) and labeled oligonucleotides insolution (either allele specific or short so as to allow sequencing byhybridization). Diagnostic tests may involve a panel of variances, oftenon a solid support, which enables the simultaneous determination of morethan one variance.

In another aspect, determining the presence of at least one kinaseactivity increasing nucleic acid variance in the erbB1 gene may entail ahaplotyping test. Methods of determining haplotypes are known to thoseof skill in the art, as for example, in WO 00/04194.

Preferably, the determination of the presence or absence of a kinaseactivity increasing nucleic acid variance involves determining thesequence of the variance site or sites by methods such as polymerasechain reaction (PCR). Alternatively, the determination of the presenceor absence of a kinase activity increasing nucleic acid variance mayencompass chain terminating DNA sequencing or minisequencing,oligonucleotide hybridization or mass spectrometry.

The methods of the present invention may be used to predict thelikelihood of effectiveness (or lack of effectiveness) of an EGFRtargeting treatment in a patient affected with or at risk for developingcancer. Preferably, cancers include cancer of epithelial origin,including, but are not limited to, gastrointestinal cancer, prostatecancer, ovarian cancer, breast cancer, head and neck cancer, lungcancer, non-small cell lung cancer, cancer of the nervous system, kidneycancer, retina cancer, skin cancer, liver cancer, pancreatic cancer,genital-urinary cancer and bladder cancer. In a preferred embodiment,the cancer is non-small cell lung cancer.

The present invention generally concerns the identification of variancesin the kinase domain of the erbB1 gene which are indicative of theeffectiveness of an EGFR targeting treatment in a patient with or atrisk for developing cancer. Additionally, the identification of specificvariances in the kinase domain of EGFR, in effect, can be used as adiagnostic or prognostic test. For example, the presence of at least onevariance in the kinase domain of erbB1 indicates that a patient willlikely benefit from treatment with an EGFR targeting compound, such as,for example, a tyrosine kinase inhibitor.

Methods for diagnostic tests are well known in the art and disclosed inpatent application WO 00/04194, incorporated herein by reference. In anexemplary method, the diagnostic test comprises amplifying a segment ofDNA or RNA (generally after converting the RNA to cDNA) spanning one ormore known variances in the kinase domain of the erbB1 gene sequence.This amplified segment is then sequenced and/or subjected topolyacrylamide gel electrophoresis in order to identify nucleic acidvariances in the amplified segment.

PCR

In one embodiment, the invention provides a method of screening forvariants in the kinase domain of the erbB1 gene in a test biologicalsample by PCR or, alternatively, in a ligation chain reaction (LCR)(see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; andNakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364), thelatter of which can be particularly useful for detecting point mutationsin the EGFR-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:675-682). The method comprises the steps of designing degenerate primersfor amplifying the target sequence, the primers corresponding to one ormore conserved regions of the gene, amplifying reaction with the primersusing, as a template, a DNA or cDNA obtained from a test biologicalsample and analyzing the PCR products. Comparison of the PCR products ofthe test biological sample to a control sample indicates variances inthe test biological sample. The change can be either and absence orpresence of a nucleic acid variance in the test biological sample.

Alternative amplification methods include: self sustained sequencereplication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (see, Kwoh, et al.,1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qb Replicase (see,Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

Primers useful according to the present invention are designed usingamino acid sequences of the protein or nucleic acid sequences of thekinase domain of the erbB1 gene as a guide, e.g. SEQ ID NO: 493, SEQ IDNO: 494, SEQ ID NO: 509, and SEQ ID NO: 510. The primers are designed inthe homologous regions of the gene wherein at least two regions ofhomology are separated by a divergent region of variable sequence, thesequence being variable either in length or nucleic acid sequence.

For example, the identical or highly, homologous, preferably at least80%-85% more preferably at least 90-99% homologous amino acid sequenceof at least about 6, preferably at least 8-10 consecutive amino acids.Most preferably, the amino acid sequence is 100% identical. Forward andreverse primers are designed based upon the maintenance of codondegeneracy and the representation of the various amino acids at a givenposition among the known gene family members. Degree of homology asreferred to herein is based upon analysis of an amino acid sequenceusing a standard sequence comparison software, such as protein-BLASTusing the default settings.

Table 3 below represents the usage of degenerate codes and theirstandard symbols:

T C A C T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC ″ TCC ″TAC TGC TTA Leu (L) TCA ″ TAA Ter TGA Ter TTG ″ TCG ″ TAG TerTGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC ″CCC ″ CAC ″ CGC ″ CTA ″ CCA ″ CAA Gln (Q) CGA ″ CTG ″ CCG ″ CAG ″ CGG ″A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC ″ ACC ″ AAC ″AGC ″ ATA ″ ACA ″ AAA Lys (K) AGA Arg (R) ATG Met (M) ACG ″ AAG ″ AGG ″G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC ″ GCC ″ GAC ″GGC ″ GTA ″ GCA ″ GAA Glu (E) GGA ″ GTG ″ GCG ″ GAG ″ GGG ″

Preferably any 6-fold degenerate codons such as L, R and S are avoidedsince in practice they will introduce higher than 6-fold degeneracy. Inthe case of L, TTR and CTN are compromised YTN (8-fold degeneracy), inthe case of R, CGN and AGR compromises at MGN (8-fold degeneracy), andfinally S, TCN and AGY which can be compromised to WSN (16-folddegeneracy). In all three cases on 6 of these will match the targetsequence. To avoid this loss of specificity, it is preferable to avoidthese regions, or to make two populations, each with the alternativedegenerate codon, e.g. for S include TCN in one pool, and AGY in theother.

Primers may be designed using a number of available computer programs,including, but not limited to Oligo Analyzer3.0; Oligo Calculator;NetPrimer; Methprimer, Primer3; WebPrimer; PrimerFinder, Primer9;Oligo2002; Pride or GenomePride; Oligos; and Codehop.

Primers may be labeled using labels known to one skilled in the art.Such labels include, but are not limited to radioactive, fluorescent,dye, and enzymatic labels.

Analysis of amplification products can be performed using any methodcapable of separating the amplification products according to theirsize, including automated and manual gel electrophoresis, massspectrometry, and the like.

Alternatively, the amplification products can be separated usingsequence differences, using SSCP, DGGE, TGGE, chemical cleavage orrestriction fragment polymorphisms as well as hybridization to, forexample, a nucleic acid arrays.

The methods of nucleic acid isolation, amplification and analysis areroutine for one skilled in the art and examples of protocols can befound, for example, in the Molecular Cloning: A Laboratory Manual(3-Volume Set) Ed. Joseph Sambrook, David W. Russel, and Joe Sambrook,Cold Spring Harbor Laboratory; 3rd edition (Jan. 15, 2001), ISBN:0879695773. Particularly useful protocol source for methods used in PCRamplification is PCR (Basics: From Background to Bench) by M. J.McPherson, S. G. Møller, R. Beynon, C. Howe, Springer Verlag; 1stedition (Oct. 15, 2000), ISBN: 0387916008.

Preferably, exons 19 and 21 of human EGFR are amplified by thepolymerase chain reaction (PCR) using the following primers: Exon19sense primer, 5′-GCAATATCAGCCTTAGGTGCGGCTC-3′ (SEQ ID NO: 505); Exon 19antisense primer, 5′-CATAGAA AGTGAACATTTAGGATGTG-3′ (SEQ ID NO: 506);Exon 21 sense primer, 5′-CTAACGTTCG CCAGCCATAAGTCC-3′ (SEQ ID NO: 507);and Exon21 antisense primer, 5′-GCTGCGAGCTCACCCAG AATGTCTGG-3′ (SEQ IDNO: 508).

In an alternative embodiment, mutations in a EGFR gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, e.g., U.S. Pat. No. 5,493,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

Other methods for detecting mutations in the EGFR gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al.,1985. Science 230: 1242. In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type EGFR sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent that cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g.,Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, etal., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the controlDNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in EGFR cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15:1657-1662. According to an exemplary embodiment, a probe based on amutant EGFR sequence, e.g., a DEL-1 through DEL-5, G719S, G857V, L883Sor L858R EGFR sequence, is hybridized to a cDNA or other DNA productfrom a test cell(s). The duplex is treated with a DNA mismatch repairenzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, e.g., U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in EGFR genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids.See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766;Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal.Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample andcontrol EGFR nucleic acids will be denatured and allowed to renature.The secondary structure of single-stranded nucleic acids variesaccording to sequence, the resulting alteration in electrophoreticmobility enables the detection of even a single base change. The DNAfragments may be labeled or detected with labeled probes. Thesensitivity of the assay may be enhanced by using RNA (rather than DNA),in which the secondary structure is more sensitive to a change insequence. In one embodiment, the subject method utilizes heteroduplexanalysis to separate double stranded heteroduplex molecules on the basisof changes in electrophoretic mobility. See, e.g., Keen, et al., 1991.Trends Genet. 7: 5.

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers,et al., 1985. Nature 313: 495. When DGGE is used as the method ofanalysis, DNA will be modified to insure that it does not completelydenature, for example by adding a GC clamp of approximately 40 bp ofhigh-melting GC-rich DNA by PCR. In a further embodiment, a temperaturegradient is used in place of a denaturing gradient to identifydifferences in the mobility of control and sample DNA. See, e.g.,Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found. See, e.g., Saiki,et al., 1986. Nature 324: 163; Saiki, et al., 1989. Proc. Natl. Acad.Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridizedto PCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology that depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization; see, e.g.,Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme3′-terminus of one primer where, under appropriate conditions, mismatchcan prevent, or reduce polymerase extension (see, e.g., Prossner, 1993.Tibtech. 11: 238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection. See, e.g., Gasparini, et al., 1992. Mol. Cell. Probes 6: 1.It is anticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification. See, e.g., Barany, 1991.Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occuronly if there is a perfect match at the 3′-terminus of the 5′ sequence,making it possible to detect the presence of a known mutation at aspecific site by looking for the presence or absence of amplification.

Solid Support and Probe

In an alternative embodiment, the detection of the presence or absenceof the at least one nucleic acid variance involves contacting a nucleicacid sequence corresponding to the desired region of the erbB1 gene,identified above, with a probe. The probe is able to distinguish aparticular form of the gene or the presence or a particular variance orvariances, e.g., by differential binding or hybridization. Thus,exemplary probes include nucleic acid hybridization probes, peptidenucleic acid probes, nucleotide-containing probes which also contain atleast one nucleotide analog, and antibodies, e.g., monoclonalantibodies, and other probes as discussed herein. Those skilled in theart are familiar with the preparation of probes with particularspecificities. Those skilled in the art will recognize that a variety ofvariables can be adjusted to optimize the discrimination between twovariant forms of a gene, including changes in salt concentration,temperature, pH and addition of various compounds that affect thedifferential affinity of GC vs. AT base pairs, such as tetramethylammonium chloride. (See Current Protocols in Molecular Biology by F. M.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, K Struhland V. B. Chanda (Editors), John Wiley & Sons.)

Thus, in preferred embodiments, the detection of the presence or absenceof the at least one variance involves contacting a nucleic acid sequencewhich includes at least one variance site with a probe, preferably anucleic acid probe, where the probe preferentially hybridizes with aform of the nucleic acid sequence containing a complementary base at thevariance site as compared to hybridization to a form of the nucleic acidsequence having a noncomplementary base at the variance site, where thehybridization is carried out under selective hybridization conditions.Such a nucleic acid hybridization probe may span two or more variancesites. Unless otherwise specified, a nucleic acid probe can include oneor more nucleic acid analogs, labels or other substituents or moietiesso long as the base-pairing function is retained.

The probe may be designed to bind to, for example, at least threecontinuous nucleotides on both sides of the deleted region of SEQ ID NO:495, SEQ ID NO: 497, or SEQ ID NO: 499. Such probes, when hybridizedunder the appropriate conditions, will bind to the variant form of EGFR,but will not bind to the wildtype EGFR.

Such hybridization probes are well known in the art (see, e.g., Sambrooket al., Eds., (most recent edition), Molecular Cloning: A LaboratoryManual, (third edition, 2001), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). Stringent hybridization conditions willtypically include salt concentrations of less than about 1M, moreusually less than about 500 mM and preferably less than about 200 mM.Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., more typically greater than about 30° C., andpreferably in excess of about 37° C. Longer fragments may require higherhybridization temperatures for specific hybridization. Other factors mayaffect the stringency of hybridization, including base composition andlength of the complementary strands, presence of organic solvents andextent of base mismatching; the combination of parameters used is moreimportant than the absolute measure of any one alone. Otherhybridization conditions which may be controlled include buffer type andconcentration, solution pH, presence and concentration of blockingreagents (e.g., repeat sequences, Cot1 DNA, blocking protein solutions)to decrease background binding, detergent type(s) and concentrations,molecules such as polymers which increase the relative concentration ofthe polynucleotides, metal ion(s) and their concentration(s),chelator(s) and their concentrations, and other conditions known ordiscoverable in the art. Formulas may be used to predict the optimalmelting temperature for a perfectly complementary sequence for a givenprobe, but true melting temperatures for a probe under a set ofhybridization conditions must be determined empirically. Also, a probemay be tested against its exact complement to determine a precisemelting temperature under a given set of condition as described inSambrook et al, “Molecular Cloning,” 3^(nd) edition, Cold Spring HarborLaboratory Press, 2001. Hybridization temperatures can be systematicallyaltered for a given hybridization solution using a support associatedwith target polynucleotides until a temperature range is identifiedwhich permits detection of binding of a detectable probe at the level ofstringency desired, either at high stringency where only targetpolynucleotides with a high degree of complementarity hybridize, or atlower stringency where additional target polynucleotides having regionsof complementarity with the probe detectably hybridize above thebackground level provided from nonspecific binding to noncomplementarytarget polynucleotides and to the support. When hybridization isperformed with potential target polynucleotides on a support under agiven set of conditions, the support is then washed under increasingconditions of stringency (typically lowered salt concentration and/orincreased temperature, but other conditions may be altered) untilbackground binding is lowered to the point where distinct positivesignals may be seen. This can be monitored in progress using a Geigercounter where the probe is radiolabeled, radiographically, using afluorescent imager, or by other means of detecting probe binding. Thesupport is not allowed to dry during such procedures, or the probe maybecome irreversibly bound even to background locations. Where a probeproduces undesirable background or false positives, blocking reagentsare employed, or different regions of the probe or different probes areused until positive signals can be distinguished from background. Onceconditions are found that provide satisfactory signal above background,the target polynucleotides providing a positive signal are isolated andfurther characterized. The isolated polynucleotides can be sequenced;the sequence can be compared to databank entries or known sequences;where necessary, full-length clones can be obtained by techniques knownin the art; and the polynucleotides can be expressed using suitablevectors and hosts to determine if the polynucleotide identified encodesa protein having similar activity to that from which the probepolynucleotide was derived. The probes can be from 10-50 nucleotides.However, much larger probes can also be employed, e.g., 50-500nucleotides or larger.

Solid Phase Support

The solid phase support of the present invention can be of any solidmaterials and structures suitable for supporting nucleotidehybridization and synthesis. Preferably, the solid phase supportcomprises at least one substantially rigid surface on whicholigonucleotides or oligonucleotide primers can be immobilized. Thesolid phase support can be made of, for example, glass, syntheticpolymer, plastic, hard non-mesh nylon or ceramic. Other suitable solidsupport materials are known and readily available to those of skill inthe art. The size of the solid support can be any of the standardmicroarray sizes, useful for DNA microarray technology, and the size maybe tailored to fit the particular machine being used to conduct areaction of the invention. Methods and materials for derivatization ofsolid phase supports for the purpose of immobilizing oligonucleotidesare known to those skill in the art and described in, for example, U.S.Pat. No. 5,919,523, the disclosure of which is incorporated herein byreference.

The solid support can be provided in or be part of a fluid containingvessel. For example, the solid support can be placed in a chamber withsides that create a seal along the edge of the solid support so as tocontain the polymerase chain reaction (PCR) on the support. In aspecific example the chamber can have walls on each side of arectangular support to ensure that the PCR mixture remains on thesupport and also to make the entire surface useful for providing theprimers.

The oligonucleotide or oligonucleotide primers of the invention areaffixed, immobilized, provided, and/or applied to the surface of thesolid support using any available means to fix, immobilize, provideand/or apply the oligonucleotides at a particular location on the solidsupport. For example, photolithography (Affymetrix, Santa Clara, Calif.)can be used to apply the oligonucleotide primers at particular positionon a chip or solid support, as described in the U.S. Pat. Nos.5,919,523, 5,837,832, 5,831,070, and 5,770,722, which are incorporatedherein by reference. The oligonucleotide primers may also be applied toa solid support as described in Brown and Shalon, U.S. Pat. No.5,807,522 (1998). Additionally, the primers may be applied to a solidsupport using a robotic system, such as one manufactured by GeneticMicroSystems (Woburn, Mass.), GeneMachines (San Carlos, Calif.) orCartesian Technologies (Irvine, Calif.).

In one aspect of the invention, solid phase amplification of targetpolynucleotides from a biological sample is performed, wherein multiplegroups of oligonucleotide primers are immobilized on a solid phasesupport. In a preferred embodiment, the primers within a group comprisesat least a first set of primers that are identical in sequence and arecomplementary to a defined sequence of the target polynucleotide,capable of hybridizing to the target polynucleotide under appropriateconditions, and suitable as initial primers for nucleic acid synthesis(i.e., chain elongation or extension). Selected primers covering aparticular region of the reference sequence are immobilized, as a group,onto a solid support at a discrete location. Preferably, the distancebetween groups is greater than the resolution of detection means to beused for detecting the amplified products. In a preferred embodiment,the primers are immobilized to form a microarray or chip that can beprocessed and analyzed via automated, processing. The immobilizedprimers are used for solid phase amplification of target polynucleotidesunder conditions suitable for a nucleic acid amplification means. Inthis manner, the presence or absence of a variety of potential variancesin the kinase domain of the erbB1 gene can be determined in one assay.

A population of target polynucleotides isolated from a healthyindividual can used as a control in determining whether a biologicalsource has at least one kinase activity increasing variance in thekinase domain of the erb1 gene. Alternatively, target polynucleotidesisolated from healthy tissue of the same individual may be used as acontrol as above.

An in situ-type PCR reactions on the microarrays can be conductedessentially as described in e.g. Embretson et al, Nature 362:359-362(1993); Gosden et al, BioTechniques 15(1):78-80 (1993); Heniford et alNuc. Acid Res. 21(14):3159-3166 (1993); Long et al, Histochemistry99:151-162 (1993); Nuovo et al, PCR Methods and Applications2(4):305-312 (1993); Patterson et al Science 260:976-979 (1993).

Alternatively, variances in the kinase domain of erbB1 can be determinedby solid phase techniques without performing PCR on the support. Aplurality of oligonucleotide probes, each containing a distinct variancein the kinase domain of erbB1, in duplicate, triplicate orquadruplicate, may be bound to the solid phase support. The presence orabsence of variances in the test biological sample may be detected byselective hybridization techniques, known to those of skill in the artand described above.

Mass Spectrometry

In another embodiment, the presence or absence of kinase activityincreasing nucleic acid variances in the kinase domain of the erbB1 geneare determined using mass spectrometry. To obtain an appropriatequantity of nucleic acid molecules on which to perform massspectrometry, amplification may be necessary. Examples of appropriateamplification procedures for use in the invention include: cloning(Sambrook et al., Molecular Cloning: A Laboratory Manual, 3^(rd)Edition, Cold Spring Harbor Laboratory Press, 2001), polymerase chainreaction (PCR) (C. R. Newton and A. Graham, PCR, BIOS Publishers, 1994),ligase chain reaction (LCR) (Wiedmann, M., et al., (1994) PCR MethodsAppl. Vol. 3, Pp. 57-64; F. Barnay Proc. Natl. Acad. Sci. USA 88, 189-93(1991), strand displacement amplification (SDA) (G. Terrance Walker etal., Nucleic Acids Res. 22, 2670-77 (1994)) and variations such asRT-PCR (Higuchi, et al., Bio/Technology 11:1026-1030 (1993)),allele-specific amplification (ASA) and transcription based processes.

To facilitate mass spectrometric analysis, a nucleic acid moleculecontaining a nucleic acid sequence to be detected can be immobilized toa solid support. Examples of appropriate solid supports include beads(e.g. silica gel, controlled pore glass, magnetic, Sephadex/Sepharose,cellulose), flat surfaces or chips (e.g. glass fiber filters, glasssurfaces, metal surface (steel, gold, silver, aluminum, copper andsilicon), capillaries, plastic (e.g. polyethylene, polypropylene,polyamide, polyvinylidenedifluoride membranes or microtiter plates)); orpins or combs made from similar materials comprising beads or flatsurfaces or beads placed into pits in flat surfaces such as wafers (e.g.silicon wafers).

Immobilization can be accomplished, for example, based on hybridizationbetween a capture nucleic acid sequence, which has already beenimmobilized to the support and a complementary nucleic acid sequence,which is also contained within the nucleic acid molecule containing thenucleic acid sequence to be detected. So that hybridization between thecomplementary nucleic acid molecules is not hindered by the support, thecapture nucleic acid can include a spacer region of at least about fivenucleotides in length between the solid support and the capture nucleicacid sequence. The duplex formed will be cleaved under the influence ofthe laser pulse and desorption can be initiated. The solid support-boundbase sequence can be presented through natural oligoribo- oroligodeoxyribonucleotide as well as analogs (e.g. thio-modifiedphosphodiester or phosphotriester backbone) or employing oligonucleotidemimetics such as PNA analogs (see e.g. Nielsen et al., Science, 254,1497 (1991)) which render the base sequence less susceptible toenzymatic degradation and hence increases overall stability of the solidsupport-bound capture base sequence.

Prior to mass spectrometric analysis, it may be useful to “condition”nucleic acid molecules, for example to decrease the laser energyrequired for volatilization and/or to minimize fragmentation.Conditioning is preferably performed while a target detection site isimmobilized. An example of conditioning is modification of thephosphodiester backbone of the nucleic acid molecule (e.g. cationexchange), which can be useful for eliminating peak broadening due to aheterogeneity in the cations bound per nucleotide unit. Contacting anucleic acid molecule with an alkylating agent such as alkyliodide,iodoacetamide, β-iodoethanol, 2,3-epoxy-1-propanol, the monothiophosphodiester bonds of a nucleic acid molecule can be transformed intoa phosphotriester bond. Likewise, phosphodiester bonds may betransformed to uncharged derivatives employing trialkylsilyl chlorides.Further conditioning involves incorporating nucleotides which reducesensitivity for depurination (fragmentation during MS) such as N7- orN9-deazapurine nucleotides, or RNA building blocks or usingoligonucleotide triesters or incorporating phosphorothioate functionswhich are alkylated or employing oligonucleotide mimetics such as PNA.

For certain applications, it may be useful to simultaneously detect morethan one (mutated) loci on a particular captured nucleic acid fragment(on one spot of an array) or it may be useful to perform parallelprocessing by using oligonucleotide or oligonucleotide mimetic arrays onvarious solid supports. “Multiplexing” can be achieved by severaldifferent methodologies. For example, several mutations can besimultaneously detected on one target sequence by employingcorresponding detector (probe) molecules (e.g. oligonucleotides oroligonucleotide mimetics). However, the molecular weight differencesbetween the detector oligonucleotides D1, D2 and D3 must be large enoughso that simultaneous detection (multiplexing) is possible. This can beachieved either by the sequence itself (composition or length) or by theintroduction of mass-modifying functionalities M1-M3 into the detectoroligonucleotide.

Preferred mass spectrometer formats for use in the invention are matrixassisted laser desorption ionization (MALDI), electrospray (ES), ioncyclotron resonance (ICR) and Fourier Transform. Methods of performingmass spectrometry are known to those of skill in the art and are furtherdescribed in Methods of Enzymology, Vol. 193: “Mass Spectrometry” (J. A.McCloskey, editor), 1990, Academic Press, New York.

Sequencing

In other preferred embodiments, determining the presence or absence ofthe at least one kinase activity increasing nucleic acid varianceinvolves sequencing at least one nucleic acid sequence. The sequencinginvolves the sequencing of a portion or portions of the kinase domain oferbB1 which includes at least one variance site, and may include aplurality of such sites. Preferably, the portion is 500 nucleotides orless in length, more preferably 100 nucleotides or less, and mostpreferably 45 nucleotides or less in length. Such sequencing can becarried out by various methods recognized by those skilled in the art,including use of dideoxy termination methods (e.g., using dye-labeleddideoxy nucleotides), minisequencing, and the use of mass spectrometricmethods.

Immunodetection

In one embodiment, determining the presence or absence of the at leastone kinase activity increasing nucleic acid variance involvesdetermining the activation state of downstream targets of EGFR.

The inventors of the present application have compared thephosphorylation status of the major downstream targets of EGFR. Forexample, the EGF-induced activation of Erk1 and Erk2, via Ras, of Aktvia PLCγ/PI3K, and of STAT3 and STAT5 via JAK2, has been examined. Erk1and Erk2, via Ras, Akt via PLCγ/PI3K, and STAT3 and STAT5 via JAK2 areessential downstream pathways mediating oncogenic effects of EGFR(R. N.Jorissen et al., Exp. Cell Res. 284, 31 (2003)).

The inventors of the present application have shown that EGF-induced Erkactivation is indistinguishable among cells expressing wild-type EGFR oreither of the two activating EGFR mutants.

In contrast, phosphorylation of both Akt and STAT5 was substantiallyelevated in cells expressing either of the mutant EGFRs. Increasedphosphorylation of STAT3 was similarly observed in cells expressingmutant EGFRs. Thus, the selective EGF-induced autophosphorylation ofC-terminal tyrosine residues within EGFR mutants is well correlated withthe selective activation of downstream signaling pathways.

In one embodiment of the present application, the presence of EGFRmutations can be determined using immunological techniques well known inthe art, e.g., antibody techniques such as immunohistochemistry,immunocytochemistry, FACS scanning, immunoblotting, radioimmunoassays,western blotting, immunoprecipitation, enzyme-linked immunosorbantassays (ELISA), and derivative techniques that make use of antibodiesdirected against activated downstream targets of EGFR. Examples of suchtargets include, for example, phosphorylated STAT3, phosphorylatedSTAT5, and phosphorylated Akt. Using phospho-specific antibodies, theactivation status of STAT3, STAT5, and Akt can be determined. Activationof STAT3, STAT5, and Akt are useful as a diagnostic indicator ofactivating EGFR mutations.

In one embodiment of the present invention, the presence of activated(phosphorylated) STAT5, STAT3, or Akt indicates that an EGFR targetingtreatment is likely to be effective.

The invention provides a method of screening for variants in the kinasedomain of the erbB1 gene in a test biological sample byimmunohistochemical or immunocytochemical methods.

Immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques,for example, may be used. IHC is the application of immunochemistry totissue sections, whereas ICC is the application of immunochemistry tocells or tissue imprints after they have undergone specific cytologicalpreparations such as, for example, liquid-based preparations.Immunochemistry is a family of techniques based on the use of a specificantibody, wherein antibodies are used to specifically target moleculesinside or on the surface of cells. The antibody typically contains amarker that will undergo a biochemical reaction, and thereby experiencea change color, upon encountering the targeted molecules. In someinstances, signal amplification may be integrated into the particularprotocol, wherein a secondary antibody, that includes the marker stain,follows the application of a primary specific antibody.

Immunohistochemical assays are known to those of skill in the art (e.g.,see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, etal., J. Cell. Biol. 105:3087-3096 (1987).

Antibodies, polyclonal or monoclonal, can be purchased from a variety ofcommercial suppliers, or may be manufactured using well-known methods,e.g., as described in Harlow et al., Antibodies: A Laboratory Manual,2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1988). In general, examples of antibodies useful in the presentinvention include anti-phospho-STAT3, anti-phospho-STAT5, andanti-phospho-Akt antibodies. Such antibodies can be purchased, forexample, from Upstate Biotechnology (Lake Placid, N.Y.), New EnglandBiolabs (Beverly, Mass.), NeoMarkers (Fremont, Calif.)

Typically, for immunohistochemistry, tissue sections are obtained from apatient and fixed by a suitable fixing agent such as alcohol, acetone,and paraformaldehyde, to which is reacted an antibody. Conventionalmethods for immunohistochemistry are described in Harlow and Lane (eds)(1988) In “Antibodies A Laboratory Manual”, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.; Ausbel et al (eds) (1987), in CurrentProtocols In Molecular Biology, John Wiley and Sons (New York, N.Y.).Biological samples appropriate for such detection assays include, butare not limited to, cells, tissue biopsy, whole blood, plasma, serum,sputum, cerebrospinal fluid, breast aspirates, pleural fluid, urine andthe like.

For direct labeling techniques, a labeled antibody is utilized. Forindirect labeling techniques, the sample is further reacted with alabeled substance.

Alternatively, immunocytochemistry may be utilized. In general, cellsare obtained from a patient and fixed by a suitable fixing agent such asalcohol, acetone, and paraformaldehyde, to which is reacted an antibody.Methods of immunocytological staining of human samples is known to thoseof skill in the art and described, for example, in Brauer et al., 2001(FASEB J, 15, 2689-2701), Smith-Swintosky et al., 1997.

Immunological methods of the present invention are advantageous becausethey require only small quantities of biological material. Such methodsmay be done at the cellular level and thereby necessitate a minimum ofone cell. Preferably, several cells are obtained from a patient affectedwith or at risk for developing cancer and assayed according to themethods of the present invention.

Other Diagnostic Methods

An agent for detecting mutant EGFR protein is an antibody capable ofbinding to mutant EGFR protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., F_(ab) or F_((ab)2)) canbe used. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently-labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently-labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect mutant EGFRmRNA, protein, or genomic DNA in a biological sample in vitro as well asin vivo. For example, in vitro techniques for detection of mutant EGFRmRNA include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of mutant EGFR protein include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of mutant EGFR genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of mutant EGFR proteininclude introducing into a subject a labeled anti-mutant EGFR proteinantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting mutant EGFR protein, mRNA,or genomic DNA, such that the presence of mutant EGFR protein, mRNA orgenomic DNA is detected in the biological sample, and comparing thepresence of mutant EGFR protein, mRNA or genomic DNA in the controlsample with the presence of mutant EGFR protein, mRNA or genomic DNA inthe test sample.

In a different embodiment, the diagnostic assay is for mutant EGFRactivity. In a specific embodiment, the mutant EGFR activity is atyrosine kinase activity. One such diagnostic assay is for detectingEGFR-mediated phosphorylation of at least one EGFR substrate. Levels ofEGFR activity can be assayed for, e.g., various mutant EGFRpolypeptides, various tissues containing mutant EGFR, biopsies fromcancer tissues suspected of having at least one mutant EGFR, and thelike. Comparisons of the levels of EGFR activity in these various cells,tissues, or extracts of the same, can optionally be made. In oneembodiment, high levels of EGFR activity in cancerous tissue isdiagnostic for cancers that may be susceptible to treatments with one ormore tyrosine kinase inhibitor. In related embodiments, EGFR activitylevels can be determined between treated and untreated biopsy samples,cell lines, transgenic animals, or extracts from any of these, todetermine the effect of a given treatment on mutant EGFR activity ascompared to an untreated control.

Method of Treating a Patient

In one embodiment, the invention provides a method for selecting atreatment for a patient affected by or at risk for developing cancer bydetermining the presence or absence of at least one kinase activityincreasing nucleic acid variance in the kinase domain of the erbB1 gene.In another embodiment, the variance is a plurality of variances, wherebya plurality may include variances from one, two, three or more geneloci.

In certain embodiments, the presence of the at least one variance isindicative that the treatment will be effective or otherwise beneficial(or more likely to be beneficial) in the patient. Stating that thetreatment will be effective means that the probability of beneficialtherapeutic effect is greater than in a person not having theappropriate presence of the particular kinase activity increasingnucleic acid variance(s) in the kinase domain of the erbB1 gene.

The treatment will involve the administration of a tyrosine kinaseinhibitor. The treatment may involve a combination of treatments,including, but not limited to a tyrosine kinase inhibitor in combinationwith other tyrosine kinase inhibitors, chemotherapy, radiation, etc.

Thus, in connection with the administration of a tyrosine kinaseinhibitor, a drug which is “effective against” a cancer indicates thatadministration in a clinically appropriate manner results in abeneficial effect for at least a statistically significant fraction ofpatients, such as a improvement of symptoms, a cure, a reduction indisease load, reduction in tumor mass or cell numbers, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treating theparticular type of disease or condition.

In a preferred embodiment, the compound is an anilinoquinazoline orsynthetic anilinoquinazoline. European. Patent Publication No. 0566226discloses anilinoquinazolines which have activity against epidermalgrowth factor (EGF) receptor tyrosine kinase. It is also known fromEuropean Patent Applications Nos. 0520722 and 0566226 that certain4-anilinoquinazoline derivatives are useful as inhibitors of receptortyrosine kinases. The very tight structure-activity relationships shownby these compounds suggests a clearly-defined binding mode, where thequinazoline ring binds in the adenine pocket and the anilino ring bindsin an adjacent, unique lipophilic pocket. Three 4-anilinoquinazolineanalogues (two reversible and one irreversible inhibitor) have beenevaluated clinically as anticancer drugs. Denny, FarmacoJanuary-February 2001; 56(1-2):51-6. Alternatively, the compound isEKB-569, an inhibitor of EGF receptor kinase (Torrance et al., NatureMedicine, vol. 6, No. 9, September 2000, p. 1024). In a most preferredembodiment, the compound is gefitinib (IRESSA®) or erlotinib (TARCEVA®).

Treatment targeting cancer cells containing at least one mutant EGFRdescribed herein may be administered alone or in combination with anyother appropriate anti-cancer treatment and/or therapeutic agent knownto one skilled in the art. In one embodiment, treatment of a pathology,such as a cancer, is provided comprising administering to a subject inneed thereof therapeutically effective amounts of a compound thatinhibits EGFR kinase activity, such as gefitinib, erlotinib, etc.,administered alone or in combination with at least one other anti-canceragent or therapy. Inhibition of activated protein kinases through theuse of targeted small molecule drugs or antibody-based strategies hasemerged as an effective approach to cancer therapy. See, e.g., G. D.Demetri et al., N. Engl. J. Med. 347, 472 (2002); B. J. Druker et al.,N. Engl. J. Med. 344, 1038 (2001); D. J. Slamon et al., N. Engl. J. Med.344, 783 (2001).

In one embodiment, the anti-cancer agent is at least onechemotherapeutic agent. In a related embodiment, the anti-cancer agentis at least one radiotherapy. In a variant embodiment, the anti-cancertherapy is an antiangiogenic therapy (e.g., endostatin, angiostatin,TNP-470, Caplostatin (Stachi-Fainaro et al., Cancer Cell 7(3), 251(2005))

The therapeutic agents may be the same or different, and may be, forexample, therapeutic radionuclides, drugs, hormones, hormoneantagonists, receptor antagonists, enzymes or proenzymes activated byanother agent, autocrines, cytokines or any suitable anti-cancer agentknown to those skilled in the art. In one embodiment, the anti-canceragent is Avastin, an anti-VEGF antibody proven successful inanti-angiogenic therapy of cancer against both solid cancers andhematological malignancies. See, e.g., Ribatti et al. 2003 J HematotherStem Cell Res. 12(1), 11-22. Toxins also can be used in the methods ofthe present invention. Other therapeutic agents useful in the presentinvention include anti-DNA, anti-RNA, radiolabeled oligonucleotides,such as antisense oligonucleotides, anti-protein and anti-chromatincytotoxic or antimicrobial agents. Other therapeutic agents are known tothose skilled in the art, and the use of such other therapeutic agentsin accordance with the present invention is specifically contemplated.

The antitumor agent may be one of numerous chemotherapy agents such asan alkylating agent, an antimetabolite, a hormonal agent, an antibiotic,an antibody, an anti-cancer biological, gleevec, colchicine, a vincaalkaloid, L-asparaginase, procarbazine, hydroxyurea, mitotane,nitrosoureas or an imidazole carboxamide. Suitable agents are thoseagents that promote depolarization of tubulin or prohibit tumor cellproliferation. Chemotherapeutic agents contemplated as within the scopeof the invention include, but are not limited to, anti-cancer agentslisted in the Orange Book of Approved Drug Products With TherapeuticEquivalence Evaluations, as compiled by the Food and Drug Administrationand the U.S. Department of Health and Human Services. Nonlimitingexamples of chemotherapeutic agents include, e.g., carboplatin andpaclitaxel. Treatments targeting EGFR kinase activity can also beadministered together with radiation therapy treatment. Additionalanti-cancer treatments known in the art are contemplated as being withinthe scope of the invention.

The therapeutic agent may be a chemotherapeutic agent Chemotherapeuticagents are known in the art and include at least the taxanes, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,triazenes; folic acid analogs, pyrimidine analogs, purine analogs, vincaalkaloids, antibiotics, enzymes, platinum coordination complexes,substituted urea, methyl hydrazine derivatives, adrenocorticalsuppressants, or antagonists. More specifically, the chemotherapeuticagents may be one or more agents chosen from the non-limiting group ofsteroids, progestins, estrogens, antiestrogens, or androgens. Even morespecifically, the chemotherapy agents may be azaribine, bleomycin,bryostatin-1, busulfan, carmustine, chlorambucil, carboplatin,cisplatin, CPT-11, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, ethinyl estradiol, etoposide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,methotrexate, methotrexate, mithramycin, mitomycin, mitotane,paclitaxel, phenyl butyrate, prednisone, procarbazine, semustinestreptozocin, tamoxifen, taxanes, taxol, testosterone propionate,thalidomide, thioguanine, thiotepa, uracil mustard, vinblastine, orvincristine. The use of any combinations of chemotherapy agents is alsocontemplated. The administration of the chemotherapeutic agent may bebefore, during or after the administration of a treatment targeting EGFRactivity.

Other suitable therapeutic agents are selected from the group consistingof radioisotope, boron addend, immunomodulator, toxin, photoactive agentor dye, cancer chemotherapeutic drug, antiviral drug, antifungal drug,antibacterial drug, antiprotozoal drug and chemosensitizing agent (See,U.S. Pat. Nos. 4,925,648 and 4,932,412). Suitable chemotherapeuticagents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and in Goodman and Gilman's ThePharmacological Basis of Therapeutics (Goodman et al., Eds. MacmillanPublishing Co., New York, 1980 and 2001 editions). Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art. Moreover a suitable therapeutic radioisotope isselected from the group consisting of α-emitters, β-emitters,γ-emitters, Auger electron emitters, neutron capturing agents that emitα-particles and radioisotopes that decay by electron capture.Preferably, the radioisotope is selected from the group consisting of225Ac, 198Au, 32P, 125I, 131I, 90Y, 186Re, 188Re, 67Cu, 177Lu, 213Bi,10B, and 211At.

Where more than one therapeutic agent is used, they may be the same ordifferent. For example, the therapeutic agents may comprise differentradionuclides, or a drug and a radionuclide. In a preferred embodiment,treatment targeting EGFR activity inhibits mutant EGFR kinase activity.

In another embodiment, different isotopes that are effective overdifferent distances as a result of their individual energy emissions areused as first and second therapeutic agents. Such agents can be used toachieve more effective treatment of tumors, and are useful in patientspresenting with multiple tumors of differing sizes, as in normalclinical circumstances.

Few of the available isotopes are useful for treating the very smallesttumor deposits and single cells. In these situations, a drug or toxinmay be a more useful therapeutic agent. Accordingly, in preferredembodiments of the present invention, isotopes are used in combinationwith non-isotopic species such as drugs, toxins, and neutron captureagents. Many drugs and toxins are known which have cytotoxic effects oncells, and can be used in connection with the present invention. Theyare to be found in compendia of drugs and toxins, such as the MerckIndex, Goodman and Gilman, and the like, and in the references citedabove.

Drugs that interfere with intracellular protein synthesis can also beused in the methods of the present invention; such drugs are known tothose skilled in the art and include puromycin, cycloheximide, andribonuclease.

The therapeutic methods of the invention may be used for cancer therapy.It is well known that radioisotopes, drugs, and toxins can be conjugatedto antibodies or antibody fragments which specifically bind to markerswhich are produced by or associated with cancer cells, and that suchantibody conjugates can be used to target the radioisotopes, drugs ortoxins to tumor sites to enhance their therapeutic efficacy and minimizeside effects. Examples of these agents and methods are reviewed inWawrzynczak and Thorpe (in Introduction to the Cellular and MolecularBiology of Cancer, L. M. Franks and N. M. Teich, eds, Chapter 18, pp.378-410, Oxford University Press. Oxford, 1986), in Immunoconjugates:Antibody Conjugates in Radioimaging and Therapy of Cancer (C. W. Vogel,ed., 3-300, Oxford University Press, N.Y., 1987), in Dillman, R. O. (CRCCritical Reviews in Oncology/Hematology 1:357, CRC Press, Inc., 1984),in Pastan et al. (Cell 47:641, 1986). in Vitetta et al. (Science238:1098-1104, 1987) and in Brady et al. (Int. J. Rad. Oncol. Biol.Phys. 13:1535-1544, 1987). Other examples of the use of immunoconjugatesfor cancer and other forms of therapy have been disclosed, inter alia,in U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744,4,460,459, 4,460,561 4,624,846, 4,818,709, 4,046,722, 4,671,958,4,046,784, 5,332,567, 5,443,953, 5,541,297, 5,601,825, 5,635,603,5,637,288, 5,677,427, 5,686,578, 5,698,178, 5,789,554, 5,922,302,6,187,287, and 6,319,500.

Additionally, the treatment methods of the invention can be used incombination with other compounds or techniques for preventing,mitigating or reversing the side effects of certain cytotoxic agents.Examples of such combinations include, e.g., administration of IL-1together with an antibody for rapid clearance, as described in e.g.,U.S. Pat. No. 4,624,846. Such administration can be performed from 3 to72 hours after administration of a primary therapeutic treatmenttargeting EGFR activity in combination with an anti-cancer agent (e.g.,with a radioisotope, drug or toxin as the cytotoxic component). This canbe used to enhance clearance of the conjugate, drug or toxin from thecirculation and to mitigate or reverse myeloid and other hematopoietictoxicity caused by the therapeutic agent.

In another aspect of the invention, cancer therapy may involve acombination of more than one tumoricidal agent, e.g., a drug and aradioisotope, or a radioisotope and a Boron-10 agent forneutron-activated therapy, or a drug and a biological response modifier,or a fusion molecule conjugate and a biological response modifier. Thecytokine can be integrated into such a therapeutic regimen to maximizethe efficacy of each component thereof.

Similarly, certain antileukemic and antilymphoma antibodies conjugatedwith radioisotopes that are β or α emitters may induce myeloid and otherhematopoietic side effects when these agents are not solely directed tothe tumor cells. This is observed particularly when the tumor cells arein the circulation and in the blood-forming organs. Concomitant and/orsubsequent administration of at least one hematopoietic cytokine (e.g.,growth factors, such as colony stimulating factors, such as G-CSF andGM-CSF) is preferred to reduce or ameliorate the hematopoietic sideeffects, while augmenting the anticancer effects.

It is well known in the art that various methods of radionuclide therapycan be used for the treatment of cancer and other pathologicalconditions, as described, e.g., in Harbert, “Nuclear Medicine Therapy”,New York, Thieme Medical Publishers, 1087, pp. 1-340. A clinicianexperienced in these procedures will readily be able to adapt thecytokine adjuvant therapy described herein to such procedures tomitigate any hematopoietic side effects thereof. Similarly, therapy withcytotoxic drugs, administered with treatment targeting EGFR activity,can be used, e.g., for treatment of cancer or other cell proliferativediseases. Such treatment is governed by analogous principles toradioisotope therapy with isotopes or radiolabeled antibodies. Theordinary skilled clinician will be able to adapt the administration ofthe additional anti-cancer therapy before, during and/or after theprimary anti-cancer therapy.

Kits

The present invention therefore also provides predictive, diagnostic,and prognostic kits comprising degenerate primers to amplify a targetnucleic acid in the kinase domain of the erbB1 gene and instructionscomprising amplification protocol and analysis of the results. The kitmay alternatively also comprise buffers, enzymes, and containers forperforming the amplification and analysis of the amplification products.The kit may also be a component of a screening, diagnostic or prognostickit comprising other tools such as DNA microarrays. Preferably, the kitalso provides one or more control templates, such as nucleic acidsisolated from normal tissue sample, and/or a series of samplesrepresenting different variances in the kinase domain of the erbB1 gene.

In one embodiment, the kit provides two or more primer pairs, each paircapable of amplifying a different region of the erbB1 gene (each regiona site of potential variance) thereby providing a kit for analysis ofexpression of several gene variances in a biological sample in onereaction or several parallel reactions.

Primers in the kits may be labeled, for example fluorescently labeled,to facilitate detection of the amplification products and consequentanalysis of the nucleic acid variances.

In one embodiment, more than one variance can be detected in oneanalysis. A combination kit will therefore comprise of primers capableof amplifying different segments of the kinase domain of the erbB1 gene.The primers may be differentially labeled, for example using differentfluorescent labels, so as to differentiate between the variances.

The primers contained within the kit may include the following primers:Exon 19 sense primer, 5′-GCAATATCAGCCTTAGGTGCGGCTC-3′ (SEQ ID NO: 505);Exon 19 antisense primer, 5′-CATAGAA AGTGAACATTTAGGATGTG-3′ (SEQ ID NO:506); Exon 21 sense primer, 5′-CTAACGTTCG CCAGCCATAAGTCC-3′ (SEQ ID NO:507); and Exon 21 antisense primer, 5′-GCTGCGAGCTCACCCAG AATGTCTGG-3′(SEQ ID NO: 508).

In a preferred embodiment, the primers are selected from the groupconsisting of SEQ ID NOS 646-673 (see Tables 5 and 6). These primershave SEQ ID NO 645 on the 5′ end of the forward primer and SEQ ID NO 674on the 5′ end of the reverse primers.

Immunodetection Kits

In further embodiments, the invention provides immunological kits foruse in detecting the activation levels of downstream EGFR targets (i.e.STAT3, STAT5, and Akt). Such kits will generally comprise one or moreantibodies that have immunospecificity for the phosphorylated form ofSTAT3, STAT5, or Akt.

A kit comprising an antibody capable of immunospecifically binding aphosphorylated protein in a mammalian cell selected from the groupconsisting of phosphorylated Akt, STAT3, and STAT5 proteins andinstructions for using the antibody to examine the mammalian cell forAkt, STAT3 or STAT5 pathway activation is provided in the presentinvention. In preferred methods, the kit comprises different antibodies,each of which is capable of immunospecifically binding phosphorylatedproteins in a mammalian cell selected from the group consisting ofphosphorylated Akt, STAT3 or STAT5 proteins.

The kit generally comprises, a) a pharmaceutically acceptable carrier;b) an antibody directed against phosphorylated STAT3, STAT5, or Akt, ina suitable container means; and c) an immunodetection reagent.Antibodies (monoclonal or polyclonal) are commercially available and mayalso be prepared by methods known to those of skill in the art, forexample, in Current Protocols in Immunology, John Wiley & Sons, Editedby: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober, 2001.

In certain embodiments, the antigen or the antibody may be bound to asolid support, such as a column matrix or well of a microtitre plate.The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with, orlinked to, the given antibody or antigen itself. Detectable labels thatare associated with or attached to a secondary binding ligand are alsocontemplated. Exemplary secondary ligands are those secondary antibodiesthat have binding affinity for the first antibody or antigen.

Suitable assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I,¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (3H) indium (^(115m)In,^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium(²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo),xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels,such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody or antigen, along witha third antibody that has binding affinity for the second antibody,wherein the third antibody is linked to a detectable label.

A number of exemplary labels are known in the art and all such labelsmay be employed in connection with the present invention. Radiolabels,nuclear magnetic spin-resonance isotopes, fluorescent labels and enzymetags capable of generating a colored product upon contact with anappropriate substrate are suitable examples.

The kits may contain antibody-label conjugates either in fullyconjugated form, in the form of intermediates, or as separate moietiesto be conjugated by the user of the kit.

The kits may further comprise a suitably aliquoted composition of anantigen whether labeled or unlabeled, as may be used to prepare astandard curve for a detection assay or as a positive control.

The kits of the invention, regardless of type, will generally compriseone or more containers into which the biological agents are placed and,preferably, suitable aliquoted. The components of the kits may bepackaged either in aqueous media or in lyophilized form.

The immunodetection kits of the invention may additionally contain oneor more of a variety of other cancer marker antibodies or antigens, ifso desired. Such kits could thus provide a panel of cancer markers, asmay be better used in testing a variety of patients. By way of example,such additional markers could include, other tumor markers such as PSA,SeLe (X), HCG, as well as p53, cyclin D1, p16, tyrosinase, MAGE, BAGE,PAGE, MUC18, CEA, p27, [bgr]HCG or other markers known to those of skillin the art.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, or even syringe or other containermeans, into which the antibody or antigen may be placed, and preferably,suitably aliquoted. Where a second or third binding ligand or additionalcomponent is provided, the kit will also generally contain a second,third or other additional container into which this ligand or componentmay be placed.

The kits of the present invention will also typically include a meansfor containing the antibody, antigen, and any other reagent containersin close confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

The methods of the present invention also encompass the identificationof compounds that interfere with the kinase activity of a variant formof the EGFR. The variant EGFR comprises at least one variance in itskinase domain. Such compounds may, for example, be tyrosine kinaseinhibitors. Methods for identifying compounds that interfere with thekinase activity of a receptor are generally known to those of skill inthe art and are further described in, for example, for example, Dhanabalet al., Cancer Res. 59:189-197 (1999); Xin et al., J. Biol. Chem.274:9116-9121 (1999); Sheu et al., Anticancer Res. 18:4435-4441;Ausprunk et al., Dev. Biol. 38:237-248 (1974); Gimbrone et al., J. Natl.Cancer Inst. 52:413-427; Nicosia et al., In vitro 18:538-549,incorporated herein by reference. In general, compounds are identified,using the methods disclosed herein, that interfere with the enhancedkinase activity characteristic of at least one variance in the kinasedomain of the erbB1 gene.

Solid Support

In another embodiment, the invention provides a kit for practicing themethods of the invention. In one embodiment, a kit for the detection ofvariances in the kinase domain of erbB1 gene on a solid support isdescribed. The kit can include, e.g. the materials and reagents fordetecting a plurality of variances in one assay. The kit can includee.g. a solid support, oligonucleotide primers for a specific set oftarget polynucleotides, polymerase chain reaction reagents andcomponents, e.g. enzymes for DNA synthesis, labeling materials, andother buffers and reagents for washing. The kit may also includeinstructions for use of the kit to amplify specific targets on a solidsupport. Where the kit contains a prepared solid support having a set ofprimers already fixed on the solid support, e.g. for amplifying aparticular set of target polynucleotides, the design and construction ofsuch a prepared solid support is described above. The kit also includesreagents necessary for conducting a PCR on a solid support, for exampleusing an in situ-type or solid phase type PCR procedure where thesupport is capable of PCR amplification using an in situ-type PCRmachine. The PCR reagents, included in the kit, include the usual PCRbuffers, a thermostable polymerase (e.g. Taq DNA polymerase),nucleotides (e.g. dNTPs), and other components and labeling molecules(e.g. for direct or indirect labeling as described above). The kits canbe assembled to support practice of the PCR amplification method usingimmobilized primers alone or, alternatively, together with solutionphase primers.

Alternatively, the kit may include a solid support with affixedoligonucleotides specific to any number of EGFR variances, furtherdefined in FIGS. 4A-4C and FIGS. 7 and 8. A test biological sample maybe applied to the solid support, under selective hybridizationconditions, for the determination of the presence or absence ofvariances in the kinase domain of erbB1.

The methods of the present invention also encompass the identificationof compounds that interfere with the kinase activity of a variant formof the EGFR. The variant EGFR comprises at least one variance in itskinase domain. However, in an alternative embodiment, the variant EGFRcomprises a secondary mutation that confers resistance to a first TKIe.g., gefitinib or erlotinib. Such compounds may, for example, betyrosine kinase inhibitors. Methods for identifying compounds thatinterfere with the kinase activity of a receptor are generally known tothose of skill in the art and are further described in, for example, forexample, Dhanabal et al., Cancer Res. 59:189-197 (1999); Xin et al., J.Biol. Chem. 274:9116-9121 (1999); Sheu et al., Anticancer Res.18:4435-4441; Ausprunk et al., Dev. Biol. 38:237-248 (1974); Gimbrone etal., J. Natl. Cancer Inst. 52:413-427; Nicosia et al., In vitro18:538-549, incorporated herein by reference. In general, compounds areidentified, using the methods disclosed herein, that interfere with theenhanced kinase activity characteristic of at least one variance in thekinase domain of the erbB1 gene. Such known variances are described inFIGS. 4, 7, 8 and Table 2.

Once identified, such compounds are administered to patients in need ofEGFR targeted treatment, for example, patients affected with or at riskfor developing cancer.

The route of administration may be intravenous (I.V.), intramuscular(I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (I.P.),intrathecal (I.T.), intrapleural, intrauterine, rectal, vaginal,topical, intratumor and the like. The compounds of the invention can beadministered parenterally by injection or by gradual infusion over timeand can be delivered by peristaltic means.

Administration may be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives. Inaddition, detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays, for example, or usingsuppositories. For oral administration, the compounds of the inventionare formulated into conventional oral administration forms such ascapsules, tablets and tonics.

For topical administration, the pharmaceutical composition (inhibitor ofkinase activity) is formulated into ointments, salves, gels, or creams,as is generally known in the art.

The therapeutic compositions of this invention are conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual.

The tyrosine kinase inhibitors useful for practicing the methods of thepresent invention are described herein. Any formulation or drug deliverysystem containing the active ingredients, which is suitable for theintended use, as are generally known to those of skill in the art, canbe used. Suitable pharmaceutically acceptable carriers for oral, rectal,topical or parenteral (including inhaled, subcutaneous, intraperitoneal,intramuscular and intravenous) administration are known to those ofskill in the art. The carrier must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects.

Formulations suitable for parenteral administration conveniently includesterile aqueous preparation of the active compound which is preferablyisotonic with the blood of the recipient. Thus, such formulations mayconveniently contain distilled water, 5% dextrose in distilled water orsaline. Useful formulations also include concentrated solutions orsolids containing the compound which upon dilution with an appropriatesolvent give a solution suitable for parental administration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Formulations for oral administration may be presented with an enhancer.Orally-acceptable absorption enhancers include surfactants such assodium lauryl sulfate, palmitoyl carnitine, Laureth-9,phosphatidylcholine, cyclodextrin and derivatives thereof; bile saltssuch as sodium deoxycholate, sodium taurocholate, sodium glycochlate,and sodium fusidate; chelating agents including EDTA, citric acid andsalicylates; and fatty acids (e.g., oleic acid, lauric acid,acylcarnitines, mono- and diglycerides). Other oral absorption enhancersinclude benzalkonium chloride, benzethonium chloride, CHAPS(3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate),Big-CHAPS(N,N-bis(3-D-gluconamidopropyl)-cholamide), chlorobutanol,octoxynol-9, benzyl alcohol, phenols, cresols, and alkyl alcohols. Anespecially preferred oral absorption enhancer for the present inventionis sodium lauryl sulfate.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods isprovided by G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers inBiology and Medicine, pp. 287-341 (Academic Press, 1979).

Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contentsof which are hereby incorporated by reference.

In one embodiment, the tyrosine kinase inhibitor of the presentinvention can be formulated into a liposome or microparticle which issuitably sized to lodge in capillary beds following intravenousadministration. When the liposome or microparticle is lodged in thecapillary beds surrounding ischemic tissue, the agents can beadministered locally to the site at which they can be most effective.Suitable liposomes for targeting ischemic tissue are generally less thanabout 200 nanometers and are also typically unilamellar vesicles, asdisclosed, for example, in U.S. Pat. No. 5,593,688 to Baldeschweiler,entitled “Liposomal targeting of ischemic tissue,” the contents of whichare hereby incorporated by reference.

Preferred microparticles are those prepared from biodegradable polymers,such as polyglycolide, polylactide and copolymers thereof. Those ofskill in the art can readily determine an appropriate carrier systemdepending on various factors, including the desired rate of drug releaseand the desired dosage.

In one embodiment, the formulations are administered via catheterdirectly to the inside of blood vessels. The administration can occur,for example, through holes in the catheter. In those embodiments whereinthe active compounds have a relatively long half life (on the order of 1day to a week or more), the formulations can be included inbiodegradable polymeric hydrogels, such as those disclosed in U.S. Pat.No. 5,410,016 to Hubbell et al. These polymeric hydrogels can bedelivered to the inside of a tissue lumen and the active compoundsreleased over time as the polymer degrades. If desirable, the polymerichydrogels can have microparticles or liposomes which include the activecompound dispersed therein, providing another mechanism for thecontrolled release of the active compounds.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier and then, if necessary,shaping the product into desired unit dosage form.

The formulations may further include one or more optional accessoryingredient(s) utilized in the art of pharmaceutical formulations, e.g.,diluents, buffers, flavoring agents, binders, surface active agents,thickeners, lubricants, suspending agents, preservatives (includingantioxidants) and the like.

Compounds of the present methods may be presented for administration tothe respiratory tract as a snuff or an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case theparticles of active compound suitably have diameters of less than 50microns, preferably less than 10 microns, more preferably between 2 and5 microns.

Generally for nasal administration a mildly acid pH will be preferred.Preferably the compositions of the invention have a pH of from about 3to 5, more preferably from about 3.5 to about 3.9 and most preferably3.7. Adjustment of the pH is achieved by addition of an appropriateacid, such as hydrochloric acid.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The kinase inhibitor of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

Predicting Mutations

In another embodiment, the present invention discloses a method topredict variances in the erbB1 gene following treatment with a tyrosinekinase inhibitor. It is generally known that response to cancertreatment with a tyrosine kinase inhibitor is often followed byresistance to that or other similar compounds. Such resistance isthought to arise through the acquisition of mutations in the drugtarget, for example in the EGFR. The ability to predict (and select)such mutations will allow for better treatment options and fewerrelapses.

In one embodiment of the present invention, DNA encoding the EGFR kinasedomain is isolated and sequenced from a tumor sample of cancer patientsthat have responded to gefitinib (or a similar EGFR targeting treatment)but have subsequently relapsed. The relapse in such patients is expectedto involve the acquisition of secondary mutations within the EGFR kinasedomain. Compounds that target, and inhibit the kinase activity of, thesenewly defined mutations are then identified using methods disclosedherein. Such compounds may be used alone, or in combination with otherknown EGFR targeting treatments, to treat cancer patients with primaryor secondary (as above) mutations in the kinase domain of EGFR.

In one embodiment, predicting variances in the kinase (catalytic) domainof the EGFR (erbB1 gene) is done in vitro. In this method, cells, e.g.fibroblast cells, are stably transfected with cDNAs containing kinasedomain mutations that have been identified in human cancer cell lines.For example, the cells may be transfected with an EGFR that bears amutation such as SEQ ID NO:495, further described in FIG. 4A, or withany number of identified or as yet unidentified kinase domain-mutatedEGFRs. The transfection of kinase domain-mutated EGFRs into cells willresult in aberrant proliferation of the cells in culture. Methods ofstable transfection are known to those of skill in the art and arefurther defined in Current Protocols in Molecular Biology by F. M.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, K. Struhland V. B. Chanda (Editors), John Wiley & Sons., 2004, incorporatedherein by reference. The transfected cells are then given an effective,yet sub-lethal, dose of a drug, preferably a tyrosine kinase inhibitor,predicted to inhibit cellular proliferation. In a preferred embodiment,the drug is an anilinoquinazoline, synthetic anilinoquinazoline,gefitinib or erlotinib. The cells are serially passaged in the presenceof drug and subclones that survive are selected. Over many generations,cells that survive (i.e. are resistant to the compound), are selectedand analyzed for variances in the erbB1 gene. Secondary variances canthus be predicted to occur following repeated treatment with a tyrosinekinase inhibitor in vivo.

Alternatively, cells are transfected with gefitinib-resistant mutantcDNA derived from human NSCLC cell lines, for example, NCI-1650 andNCI-1975. Each cell line has a heterozygous mutation with the kinasedomain of EGFR, and is, therefore, expected to be sensitive togefitinib. The EGFR mutation in NCI-1650 consists of an in-framedeletion of 15 nucleotides at position 2481-2495 of SEQ ID NO: 211(delLE746-A750) within exon 19, while NCI-1975 has a missense mutationwithin exon 21 that substitutes a G for T at nucleotide 2818 of SEQ IDNO: 511 (L858R). As shown herein, the L858R mutation in NCI-H1975 isactivating and confers increased sensitivity to gefitinib in vitro.Other cancer cell lines that harbor EGFR kinase domain mutations may beutilized. The cancer cell lines may include lung cancer as well as othercancers that are found to harbor such mutations.

The cells may be treated with a mutagen in order to increase thefrequency with which cells acquire secondary mutations. A mutagen mayinduce mutations at different frequencies depending upon the dosageregimen, mode of delivery, and the developmental stage of the organismor cell upon mutagen administration, all parameters of which aredisclosed in the prior art for different mutagens or mutagenesistechniques. The mutagen may be an alkylating agent, such as ethylmethanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU) orN-methyl-N-nitrosourea (MNU). Alternatively, the mutagen may be, forexample, phocarbaxine hydrochloride (Prc), methyl methanesulfonate(MeMS), chlorambucil (Chl), melphalan, porcarbazine hydrochloride,cyclophosphamide (Cp), diethyl sulfate (Et₂SO₄), acrylamide monomer(AA), triethylene melamin (TEM), nitrogen mustard, vincristine,dimethylnitrosamine, N-methyl-N′-nitro-Nitrosoguanidine (MNNG), 7,12dimethylbenz(a)anthracene (DMBA), ethylene oxide,hexamethylphosphoramide, bisulfan, and ethyl methanesulforate (EtMs).Methods of treating cells with mutagens is described, for example, inU.S. Pat. No. 6,015,670, incorporated herein by reference. Followingmutagenesis, cells (i.e. transfected with variant EGFR or human cancercell line derived) can be cultured in gefitinib-supplemented medium toselect for the outgrowth of resistant clones. Subcultivation ofindividual clones can be followed, for example, by nucleotide sequencedetermination of the EGFR gene following specific PCR-mediatedamplification of genomic DNA corresponding to the EGFR kinase domain.

In another embodiment, cells (with an EGFR variance) are seriallypassaged in the presence of gradually increasing concentrations ofgefitinib (or a similar tyrosine kinase inhibitor) over a course ofseveral weeks or months in order to select for the spontaneousacquisition of mutations within the EGFR gene that confer resistance togefitinib. Selected cells (that continue to proliferate at relativelyhigh gefitinib concentration) can be isolated as colonies, and mutationswill be identified as described above. Such variances can thus bepredicted to occur following repeated treatment with a tyrosine kinaseinhibitor in vivo. See, for example, Scappini et al., Cancer, Apr. 1,2004, Vol. 100, pg. 1459, incorporated herein by reference.

In yet another embodiment, a variant form of the EGFR gene can bepropagated in a DNA repair-deficient bacterial strain beforere-introducing it into stably selected cell lines. Replication in suchbacteria will enhance the frequency of mutagenesis. Alternatively,“error-prone” PCR can be utilized to enhance the frequency of mutationsin the cloned EGFR DNA in vitro, using standard methods, known to thoseof skill in the art.

In another embodiment, predicting variances in the kinase domain of theerbB1 gene is done in vivo. For example, a kinase activity increasingvariant form of the erbB1 gene is transfected into an animal, i.e. amouse, generating a cancer model. The animal is then treated with aneffective dose of a compound, preferably an anilinoquinazoline,synthetic anilinoquinazoline, gefitinib or erlotinib. Upon repeatedexposure to the compound, the cancer is initially inhibited. As inhumans treated with such compounds, tumor cells in the animal acquiremutations which make them resistant to such treatment. The methods ofthe present invention allow for the isolation and characterization ofthe erbB1 gene in such resistant tumors. Compounds that specificallytarget these newly characterized variances are useful in the treatmentof patients suspected of carrying such a mutated erbB1 gene. Suchpatients include, for example, patients who initially respond to therapywith a tyrosine kinase inhibitor, but subsequently fail to respond tothe same or similar compound.

Methods of creating an animal model are known to those of skill in theart and are further defined in e.g., Ohashi et al., Cell, 65:305-317(1991); Adams et al., Nature, 325:223-228 (1987); and Roman et al.,Cell, 61:383-396 (1990)., incorporated herein by reference. In the caseof fertilized oocytes, the preferred method of transgene introduction isby microinjection, see, e.g., Leder et al., U.S. Pat. Nos. 4,736,866 and5,175,383, which are incorporated herein by reference, whereas in thecase of embryonic stem (ES) cells, the preferred method iselectroporation. However, other methods including viral delivery systemssuch as retroviral infection, or liposomal fusion can be used. Theisolation and characterization of nucleic acid is described above and inthe examples.

The above-identified kinase activity increasing variances in the erbB1gene may be screened for in patients (diagnostically or prognostically),using the methods of the present invention. The presence or absence ofsuch mutations may then be used as a criteria for determining onessensitivity to treatment with an EGFR targeting compound, such as, forexample, a tyrosine kinase inhibitor.

Compounds that specifically target these newly defined variances,whether detected in vivo or in vitro, can be selected using techniquesknown in the art and discussed herein. Candidate drug screening assaysmay be used to identify bioactive candidate agents that inhibit theactivity of variant forms of EGFR. Of particular interest are screeningassays for agents that have a low toxicity for human cells. A widevariety of assays may be used for this purpose, including labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, enzyme activity assays, immunoassays for protein binding, andthe like. The purified mutant EGFR protein may also be used fordetermination of three-dimensional crystal structure, which can be usedfor modeling intermolecular interactions, transporter function, etc.Such compounds may be, for example, tyrosine kinase inhibitors,antibodies, aptamers, siRNAs, and vectors that inhibit the kinaseactivity of EGFR.

In another embodiment, compounds useful in the method of the presentinvention are antibodies which interfere with kinase signaling via themutant EGFR, including monoclonal, chimeric humanized, and recombinantantibodies and fragment thereof which are characterized by their abilityto inhibit the kinase activity of the EGFR and which have low toxicity.

Neutralizing antibodies are readily raised in animals such as rabbits ormice by immunization with an EGFR with at least one nucleic acidvariance in its kinase domain. Immunized mice are particularly usefulfor providing sources of B cells for the manufacture of hybridomas,which in turn are cultured to produce large quantities of anti-EGFRmonoclonal antibodies. Chimeric antibodies are immunoglobin moleculescharacterized by two or more segments or portions derived from differentanimal species. Generally, the variable region of the chimeric antibodyis derived from a non-human mammalian antibody, such as murinemonoclonal antibody, and the immunoglobin constant region is derivedfrom a human immunoglobin molecule. Preferably, both regions and thecombination have low immunogenicity as routinely determined. Humanizedantibodies are immunoglobin molecules created by genetic engineeringtechniques in which the murine constant regions are replaced with humancounterparts while retaining the murine antigen binding regions. Theresulting mouse-human chimeric antibody should have reducedimmunogenicity and improved pharmacokinetics in humans. Preferredexamples of high affinity monoclonal antibodies and chimeric derivativesthereof, useful in the methods of the present invention, are describedin the European Patent Application EP 186,833; PCT Patent Application WO92/16553; and U.S. Pat. No. 6,090,923.

Existing or newly identified compounds as described above are useful inthe treatment of patients carrying primary and/or secondary EGFRmutations.

In a preferred embodiment, the compound is an inhibitor of the tyrosinekinase activity of an EGFR with at least one variance in its kinasedomain, particularly small molecule inhibitors having selective actionon “mutated” EGFRs as compared to other tyrosine kinases. Inhibitors ofEGFR include, but are not limited to, tyrosine kinase inhibitors such asquinazolines, such as PID 153035, 4-(3-chloroanilino) quinazoline, orCP-358,774, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines (Traxler et al., (1996) J.Med Chem 39:2285-2292), curcumin (diferuloyl methane) (Laxmin arayana,et al., (1995), Carcinogen 16:1741-1745), 4,5-bis(4-fluoroanilino)phthalimide (Buchdunger et al. (1995) Clin. Cancer Res. 1:813-821;Dinney et al. (1997) Clin. Cancer Res. 3:161-168); tyrphostinscontaining nitrothiophene moieties (Brunton et al. (1996) Anti CancerDrug Design 11:265-295); the protein kinase inhibitor ZD-1 839(AstraZeneca); CP-358774 (Pfizer, Inc.); PD-01 83805 (Warner-Lambert),EKB-569 (Torrance et al., Nature Medicine, Vol. 6, No. 9, September2000, p. 1024), HKI-272 and HKI-357 (Wyeth); or as described inInternational patent application W099/09016 (American Cyanamid);W098/43960 (American Cyanamid); W097/38983 (Warener Labert); W099/06378(Warner Lambert); W099/06396 (Warner Lambert); W096/30347 (Pfizer,Inc.); W096/33978 (Zeneca); W096/33977 (Zeneca); and W096/33980) Zeneca;all herein incorporated by reference.

In another embodiment, an antisense strategy may be used to interferewith the kinase activity of a variant EGFR. This approach may, forinstance, utilize antisense nucleic acids or ribozymes that blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme. For a generaldiscussion of antisense technology, see, e.g., Antisense DNA and RNA,(Cold Spring Harbor Laboratory, D. Melton, ed., 1988).

Reversible short inhibition of variant EGFR gene transcription may alsobe useful. Such inhibition can be achieved by use of siRNAs. RNAinterference (RNAi) technology prevents the expression of genes by usingsmall RNA molecules such as small interfering RNAs (siRNAs). Thistechnology in turn takes advantage of the fact that RNAi is a naturalbiological mechanism for silencing genes in most cells of many livingorganisms, from plants to insects to mammals (McManus et al., NatureReviews Genetics, 2002, 3(10) p. 737). RNAi prevents a gene fromproducing a functional protein by ensuring that the moleculeintermediate, the messenger RNA copy of the gene is destroyed. siRNAscan be used in a naked form and incorporated in a vector, as describedbelow. One can further make use of aptamers to specifically inhibitvariant EGFR gene transcription, see, for example, U.S. Pat. No.6,699,843. Aptamers useful in the present invention may be identifiedusing the SELEX process. The methods of SELEX have been described in,for example, U.S. Pat. Nos. 5,707,796, 5,763,177, 6,011,577, 5,580,737,5,567,588, and 5,660,985.

An “antisense nucleic acid” or “antisense oligonucleotide” is a singlestranded nucleic acid molecule, which, on hybridizing under cytoplasmicconditions with complementary bases in a RNA or DNA molecule, inhibitsthe latter's role. If the RNA is a messenger RNA transcript, theantisense nucleic acid is a countertranscript or mRNA-interferingcomplementary nucleic acid. As presently used, “antisense” broadlyincludes RNA-RNA interactions, RNA-DNA interactions, ribozymes, RNAi,aptamers and Rnase-H mediated arrest.

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these ribozymes,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1989,Science 245(4915) p. 276). Because they are sequence-specific, onlymRNAs with particular sequences are inactivated.

Antisense nucleic acid molecules can be encoded by a recombinant genefor expression in a cell (e.g., U.S. Pat. Nos. 5,814,500; 5,811,234), oralternatively they can be prepared synthetically (e.g., U.S. Pat. No.5,780,607).

The present invention further provides methods of treating patients withcancer. In particular, patients with at least one nucleic acid variancein the kinase domain of EGFR. The treatment method comprisesadministering an siRNA-containing composition to a patient within anappropriate time window. The siRNAs may be chemically synthesized,produced using in vitro transcription, etc. In addition, the siRNAmolecule can be customized to individual patients in such a way as tocorrespond precisely to the mutation identified in their tumor. SincesiRNA can discriminate between nucleotide sequences that differ by onlya single nucleotide, it is possible to design siRNAs that uniquelytarget a mutant form of the EGFR gene that is associated with either asingle nucleotide substitution or a small deletion of severalnucleotides—both of which have been identified in tumors as describedherein. SiRNAs have been described in Brummelkamp et al., Science 296;550-553, 2002, Jaque et al., Nature 418; 435-438, 2002, Elbashir S. M.et al. (2001) Nature, 411: 494-498, McCaffrey et al. (2002), Nature,418: 38-39; Xia H. et al. (2002), Nat. Biotech. 20: 1006-1010, Novina etal. (2002), Nat. Med. 8: 681-686, and U.S. Application No. 20030198627.

An important advantage of such a therapeutic strategy relative to theuse of drugs such as gefitinib, which inhibit both the mutated receptorand the normal receptor, is that siRNA directed specifically against themutated EGFR should not inhibit the wildtype EGFR. This is significantbecause it is generally believed that the “side effects” of gefitinibtreatment, which include diarrhea and dermatitis, are a consequence ofinhibition of EGFR in normal tissues that require its function.

The delivery of siRNA to tumors can potentially be achieved via any ofseveral gene delivery “vehicles” that are currently available. Theseinclude viral vectors, such as adenovirus, lentivirus, herpes simplexvirus, vaccinia virus, and retrovirus, as well as chemical-mediated genedelivery systems (for example, liposomes), or mechanical DNA deliverysystems (DNA guns). The oligonucleotides to be expressed for suchsiRNA-mediated inhibition of gene expression would be between 18 and 28nucleotides in length.

In another embodiment, the compounds are antisense molecules specificfor human sequences coding for an EGFR having at least one variance inits kinase domain. The administered therapeutic agent may be anantisense oligonucleotides, particularly synthetic oligonucleotides;having chemical modifications from native nucleic acids, or nucleic acidconstructs that express such anti-sense molecules as RNA. The antisensesequence is complementary to the mRNA of the targeted EGFR genes, andinhibits expression of the targeted gene products (see e.g. Nyce et al.(1997) Nature 385:720). Antisense molecules inhibit gene expression byreducing the amount of mRNA available for translation, throughactivation of RNAse H or steric hindrance. One or a combination ofantisense molecules may be administered, where a combination maycomprise multiple different sequences from a single targeted gene, orsequences that complement several different genes.

A preferred target gene is an EGFR with at least one nucleic acidvariance in its kinase domain. The gene sequence is incorporated herein,such as, for example, in FIG. 5. Generally, the antisense sequence willhave the same species of origin as the animal host.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where the vector isintroduced and expressed in the targeted cells. The transcriptionalinitiation will be oriented such that the antisense strand is producedas an RNA molecule.

The anti-sense RNA hybridizes with the endogenous sense strand mRNA,thereby blocking expression of the targeted gene. The nativetranscriptional initiation region, or an exogenous transcriptionalinitiation region may be employed. The promoter may be introduced byrecombinant methods in vitro, or as the result of homologous integrationof the sequence into a chromosome. Many strong promoters that are activein muscle cells are known in the art, including the O-actin promoter,SV40 early and late promoters, human cytornegalovirus promoter,retroviral LTRs, etc. Transcription vectors generally have convenientrestriction sites located near the promoter sequence to provide for theinsertion of nucleic acid sequences. Transcription cassettes maybeprepared comprising a transcription initiation region, the target geneor fragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained incells, usually for a period of at least about one day, more usually fora period of at least about several days.

Aptamers are also useful. Aptamers are a promising new class oftherapeutic oligonucleotides or peptides and are selected in vitro tospecifically bind to a given target with high affinity, such as forexample ligand receptors. Their binding characteristics are likely areflection of the ability of oligonucleotides to form three dimensionalstructures held together by intramolecular nucleobase pairing. Aptamersare synthetic DNA, RNA or peptide sequences which may be normal andmodified (e.g. peptide nucleic acid (PNA), thiophosphorylated DNA, etc)that interact with a target protein, ligand (lipid, carbohydrate,metabolite, etc). In a further embodiment, RNA aptamers specific for avariant EGFR can be introduced into or expressed in a cell as atherapeutic.

Peptide nucleic acids (PNAs) are compounds that in certain respects aresimilar to oligonucleotides and their analogs and thus may mimic DNA andRNA. In PNA, the deoxyribose backbone of oligonucleotides has beenreplaced by a pseudo-peptide backbone (Nielsen et al. 1991 Science 254,1457-1500). Each subunit, or monomer, has a naturally occurring ornon-naturally occurring nucleobase attached to this backbone. One suchbackbone is constructed of repeating units of N-(2-aminoethyl) glycinelinked through amide bonds. PNA hybridises with complementary nucleicacids through Watson and Crick base pairing and helix formation. ThePseudo-peptide backbone provides superior hybridization properties(Egholm et al. Nature (1993) 365, 566-568), resistance to enzymaticdegradation (Demidov et al. Biochem. Pharmacol. (1994) 48, 1310-1313)and access to a variety of chemical modifications (Nielsen and HaaimaChemical Society Reviews (1997) 73-78). PNAs specific for a variant EGFRcan be introduced into or expressed in a cell as a therapeutic. PNAshave been described, for example, in U.S. Application No. 20040063906.

Patients to be treated with a compound which targets a variant EGFRinclude, for example, patients diagnosed with a primary or secondarymutation in their EGFR, patients who initially respond to therapy with atyrosine kinase inhibitor, but subsequently fail to respond to the sameor similar compound. Alternatively, compounds that target secondary EGFRmutations may be given to cancer patients in combination with compoundsthat target primary EGFR mutations, for example, gefitinib, as acombination therapy. By combining compounds that target both primary andsecondary EGFR mutations, the likelihood of resistance will be reduced.

Additional EGFR mutations that confer resistance to currently knownanti-cancer therapeutics, including but not limited to EGFR tyrosinekinase inhibitors gefitinib, erlotinib and the like, are within thescope of the invention. Resistant EGFR mutants are predicted to havemutants analogous to mutants identified in kinase domains of relatedtyrosine kinase domain containing proteins that have high homology inthis kinase region. Papers describing mutations in analogous proteinsinclude those known in the art for BCR-ABL. See, e.g., Bradford et al.Blood. 2003 Jul. 1; 102(1):276-83, Epub 2003 Mar. 6; Hochhaus et al.,Leukemia. 2002 November; 16(11):2190-6; and Al-Ali et al., Hematol J.2004; 5(1):55-60.

A mutant EGFR resistant to known EGFR tyrosine kinase inhibitorsincludes any one or more EGFR polypeptides, or a nucleotide encoding thesame, with a non-wild type residue at one or more positions analogous toc-ab1 (BCR-ABL) residues that confirm an imatinib resistant phenotype.The residues that when mutated in EGFR confer drug resistance includeespecially those residues from the kinase domain, including but notlimited to, e.g., the P-loop and the activation loop, wherein themutated residues in the EGFR polypeptide are analogous to c-ableresidues. Contemplated resistant EGFR mutants have non-wild typeresidues at the amino acids positions that are analogous to at leastpositions Met 244, Leu 248, Gly 250, Gln 252, Tyr 253, Glu 255, Asp 276,Thr 315, Phe 317, Met 351, Glu 355, Phe 359, His 396, Ser 417, and Phe486 of BCR-ABL, see, for example Table S3C and FIG. 9. These BCL-ABLresidues correspond to residues Lys 714, Leu 718, Ser 720, Ala 722, Phe723, Thr 725, Ala 750, Thr 790, Leu 792, Met 825, Glu 829, Leu 833, His870, Thr 892, Phe 961, respectively, in EGFR. See, e.g., Table S3C, FIG.9.

Prognostic Testing

The methods of the present invention are used as a prognostic indicatorof the development of cancer. Alternatively, the methods are used todetect cancer that is present but has not yet been diagnosed or is at astage that is undetectable. Patients at risk for developing cancer arescreened, using the methods of the present invention, for the presenceof kinase activity increasing nucleic acid variation in the erbB1 gene.The presence of a variance or variances in the kinase domain of theerbB1 gene indicate the presence or imminent presence of cancer. Thus,the presence of variances in the kinase domain of the erbB1 gene suggestthat a patient would benefit from an EGFR targeted treatment. Asdescribed herein, an EGFR targeted treatment is preferably treatmentwith a tyrosine kinase inhibitor.

In a preferred embodiment of the present invention, a patient isscreened for the presence or absence of nucleic acid variances in thekinase domain of the erbB1 gene by obtaining a biological sample. Thesample may be any sample from the patient including tissue, e.g., fromthe tongue, mouth, cheek, trachea, bronchial tube, lungs, etc. or fluid,e.g., from sputum or lung aspirates. Methods of obtaining thesebiological specimens are well known to those of skill in the art.

Thus, the invention provides a method for identifying a disease ordisorder associated with aberrant mutant EGFR expression or activity inwhich a test sample is obtained from a subject and mutant EGFR proteinor nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein thepresence of mutant EGFR protein or nucleic acid is diagnostic for asubject having or at risk of developing a disease or disorder associatedwith aberrant mutant EGFR expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue, especially a tissue biopsy sample.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant mutant EGFR expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a disorder. Thus, the inventionprovides methods for determining whether a subject can be effectivelytreated with an agent for a disorder associated with aberrant mutantEGFR expression or activity in which a test sample is obtained andmutant EGFR protein or nucleic acid is detected (e.g., wherein thepresence of mutant EGFR protein or nucleic acid is diagnostic for asubject that can be administered the agent to treat a disorderassociated with mutant EGFR expression or activity).

EXAMPLES Example 1 Nucleotide Sequence Analysis of Tumor Specimens

Tumor specimens from initial diagnostic or surgical procedures werecollected from patients with NSCLC who were subsequently treated withGefitinib, under an IRB-approved protocol. Frozen tumor specimens, alongwith matched normal tissue, were available for four cases, andparaffin-embedded material was used for the remaining specimens. Inaddition, 25 unselected cases of primary NSCLC (15 bronchioalveolar, 7adenocarcinoma, and 3 large cell lung cancers), with matched normaltissues, were obtained from the Massachusetts General Hospital tumorbank. For mutational analysis of the entire EGFR coding sequence, DNAwas extracted from specimens, followed by amplification of all 28 exons,automated sequencing of uncloned PCR fragments, and analysis ofelectropherograms in both sense and antisense direction for the presenceof heterozygous mutations. All sequence variants were confirmed bymultiple independent PCR amplifications. Primer sequences andamplification conditions are provided in Supplementary Material. EGFRmutations in exons 19 and 21 were also sought in primary tumors of thebreast (15 cases), colon (20 cases), kidney (1-6 cases), and brain (4cases), along with a panel of 78 cancer-derived cell lines representingdiverse histologies (listed below).

Functional Analysis of Mutant EGFR Constructs

The L858R and delL747-P753insS mutations were introduced into the fulllength EGFR coding sequence using site-directed mutagenesis and insertedinto a cytomegalovirus-driven expression construct (pUSE, Upstate).Cos-7 cells were transfected (Lipofectamine 2000, Invitrogen) using 1 μgof the expression constructs, followed after 18 hrs by replating at5×10⁴ cells/well (12-well plates, Costar) in DMEM lacking fetal calfserum. After 16 hrs of serum starvation, cells were stimulated with 10ng/ml of EGF (SIGMA). To demonstrate Gefitinib inhibition, the drug wasadded to the culture medium 3 hrs prior to the addition of EGF (30 minstimulation with 100 ng/ml of EGF). Cell lysates were prepared in 100 μLof Laemmli lysis buffer, followed by resolution of proteins on 10%SDS-PAGE, transfer to PVDF membranes, and Western blot analysis usingenhanced chemiluminescence reagent (Amersham). Autophosphorylation ofEGFR was measured using antibody to phosphotyrosine Y-1068, andcomparable protein expression was shown using anti-EGFR antibody(working concentration of 1:1000; Cell Signaling Technology).

Mutational Analysis

The polymerase chain reaction was used to amplify the 28 exonscomprising the EGFR gene using DNA isolated from primary tumor tissue ortumor-derived cell-lines. Primer pairs used were: Exon 1,CAGATTTGGCTCGACCTGGACATAG (sense) (SEQ ID NO: 513) andCAGCTGATCTCAAGGAAACAGG (antisense) (SEQ ID NO: 514); Exon 2,GTATTATCAGTCAC TAAAGCTCAC (sense) (SEQ ID NO: 515) andCACACTTCAAGTGGAATTCTGC (SEQ ID NO: 516); Exon 3, CTCGTGTGCATTAGGGTTCAACTGG (sense) (SEQ ID NO: 517) andCCTTCTCCGAGGTGGAATTGAGTGAC (antisense) (SEQ ID NO: 518); Exon 4,GCTAATTGCGGGACTCTTGTTCGCAC (sense) (SEQ ID NO: 519) and TACATGCTTTTCTAGTGGTCAG (antisense) (SEQ ID NO: 520); Exon 5,GGTCTCAAGTGATTCTACAAACCAG (sense) (SEQ ID NO: 521) andCCTTCACCTACTGGTTCACATCTG (antisense) (SEQ ID NO: 522); Exon 6, CATGGTTTGACTTAGTTTGAATGTGG (sense) (SEQ ID NO: 523) andGGATACTAAAGATACTITGTCAC CAGG (antisense) (SEQ ID NO: 524); Exon 7,GAACACTAGGCTGCAAAGACAGTAAC (sense) (SEQ ID NO: 525) andCCAAGCAAGGCAAACACATCCACC (antisense) (SEQ ID NO: 526); Exon 8,GGAGGATGGAGCC TTTCCATCAC (sense) (SEQ ID NO: 527) andGAAGAGGAAGATGTGTTCCTTTGG (antisense) (SEQ ID NO: 528); Exons 9 and 10,GAATGAAGGATGATGTGGCAGTGG (sense) (SEQ ID NO: 529) and CAAAACATCAGCCATTAACGG (antisense) (SEQ ID NO: 530); Exon 11,CCACTTACTGTTCATATAATACAGAG (sense) (SEQ ID NO: 531) andCATGTGAGATAGCATFTGGGAATGC (antisense) (SEQ ID NO: 532); Exon 12,CATGACCT ACCATCATTGGAAAGCAG (sense) (SEQ ID NO: 533) andGTAATTTCACAGTTAGGAATC (sense) (SEQ ID NO: 534); Exon 13,GTCACCCAAGGTCATGGAGCACAGG (sense) (SEQ ID NO: 535) and CAGAATGCCTGTAAAGCTATAAC (antisense) (SEQ ID NO: 536); Exon 14,GTCCTGGAGTCCCAACTCCTTGAC (sense) (SEQ ID NO: 537) and GGAAGTGGCTCTGATGGCCGTCCTG (antisense) (SEQ ID NO: 538); Exon 15, CCACTCACACACACTAAATATTTTAAG (sense) (SEQ ID NO: 539) andGACCAAAACACCTTAAGTAA CTGACTC (antisense) (SEQ ID NO: 540); Exon 16, CCAATCCAACATCCAGACACATAG (sense) (SEQ ID NO: 541) andCCAGAGCCATAGAAACTTGATCAG (antisense) (SEQ ID NO: 542); Exon 17,GTATGGACTATGGC ACTTCAATTGCATGG (sense) (SEQ ID NO: 543) andCCAGAGAACATGGCAACCAGCACAGGAC (antisense) (SEQ ID NO: 544); Exon 18,CAAATGAGCTGGCAAGTGCCGTGTC (sense) (SEQ ID NO: 545) and GAGTTTCCCAAACACTCAGTGAAAC (antisense) (SEQ ID NO: 546) orCAAGTGCCGTGTCCTGGCACCCAAGC (sense) (SEQ ID NO: 675) andCCAAACACTCAGTGAAACAAAGAG (antisense) (SEQ ID NO: 676); Exon 19,GCAATATCAGCC TTAGG TGCGGCTC (sense) (SEQ ID NO: 547) andCATAGAAAGTGAACATrTAGGATGTG (antisense) (SEQ ID NO: 548); Exon 20,CCATGAGTACGTATTTTGAAACTC (sense) (SEQ ID NO: 549) and CATATCC CCATGGCAAACTCTTGC (antisense) (SEQ ID NO: 550); Exon 21, CTAACGTTCGCCAGCCATAAGTCC (sense) (SEQ ID NO: 551) and GCTGCGAGCTCACCCAGAATGTCTGG(antisense) (SEQ ID NO: 552); Exon 22, GACGGG TCCTGGGGTGATCTGGCTC(sense) (SEQ ID NO: 553) and CTCAGTACAATAGATAGACAGCAATG (antisense) (SEQID NO: 684); Exon 23, CAGGACTACAGAAATGTAGGTTTC (sense) (SEQ ID NO: 555)and GTGCCTG CCTTAAGTAATGTGATGAC (antisense) (SEQ ID NO: 556); Exon 24,GACTGG AAGTGTCGCA TCACCAATG (sense) (SEQ ID NO: 557) andGGTTTAATAATGCGATCTGGGACAC (antisense) (SEQ ID NO: 558); Exon 25,GCAGCTATAATITAGAGAACCAAGG (sense) (SEQ ID NO: 559) and GGTT AAAATTGACTTCATTTCCATG (antisense) (SEQ ID NO: 560); Exon 26, CCTAGTFGCTCTAAA ACTAACG(sense) (SEQ ID NO: 561) and CTGTGAGGCGTGACAGCCGTGCAG (antisense) (SEQID NO: 562); Exon 27, CAACCTACTAATCAG AACCAGCATC (sense) (SEQ ID NO:563) and CCTTCACTGTGTCTGC AAATCTGC (antisense) (SEQ ID NO: 564); Exon28, CCTGTCATAAGTCTCCTTGTTGAG (sense) (SEQ ID NO: 565) andCAGTCTGTGGGTCTAAG AGCTAATG (antisense) (SEQ ID NO: 566). Annealingtemperatures were 58° C. (exons 1, 3, 4, 7-10, 12-25, 27, and 28), 56°C. (exons 2, 5, 6, and 26), or 52° C. (exon 11).

Nested PCR amplification of DNA extracted from archival tumor tissue wasperformed as follows. An initial PCR for exons 2, 5, 6, 7, 11, 12, 14,16, 18, 19, 20, 21, 23, 24, 25, 26, and 27 was generated using primersand conditions described above. Subsequently, 2 μl of this reaction wasamplified in a secondary PCR using the following internal primer pairs:Exon 2, CAGGAATGGGTGAGTCTCTGTGTG (sense) (SEQ ID NO: 567) andGTGGAATTCTGCCCAGGCCTTTC (antisense) (SEQ ID NO: 568); Exon 5,GATTCTACAAACCA GCCAGCCAAAC (sense) (SEQ ID NO: 569) andCCTACTGGTTCACATCTGACCCTG (antisense) (SEQ ID NO: 570); Exon 6,GTTTGAATGTGGTITCGTTGGAAG (sense) (SEQ ID NO: 571) and CTTTGTCACCAGGCAGAGG GCAATATC (antisense) (SEQ ID NO: 572); Exon 7,GACAGTAACTTGGGCTITCTGAC (sense) (SEQ ID NO: 573) andCATCCACCCAAAGACTCTCCAAG (antisense) (SEQ ID NO: 574); Exon11, CTGTTCATATAATAC AGAGTCCCTG (sense) (SEQ ID NO: 575) and GAGAGATGCAGGAGCTCTGTGC(antisense) (SEQ ID NO: 576); Exon12, GCAGTTTGTAGTCAATCAAAGGTGG (sense)(SEQ ID NO: 577) and GTAAITTAAATGGGAAT AGCCC (antisense) (SEQ ID NO:578); Exon14, CAACTCCTTGACCATTACCTCAAG (sense) (SEQ ID NO: 579) andGATGGCCGTCCTGCCCACACAGG (antisense) (SEQ ID NO: 580); Exon16,GAGTAGTTTAGCA TATATTGC (sense) (SEQ ID NO: 581) andGACAGTCAGAAATGCAGGAAAGC (antisense) (SEQ ID NO: 582); Exon18,CAAGTGCCGTGTCCTGGCACCCAAGC (sense) (SEQ ID NO: 583) and CCAAACACTCAGTGAAACAAAGAG (antisense) (SEQ ID NO: 584) or GCACCCAAGCCCATGCCGTGGCTGC(sense) (SEQ ID NO: 677) and GAAACAAAGAGTAAAGTAGATGATGG (antisense) (SEQID NO: 678); Exon 19, CCTTAGGTGCGGCTCCACAGC (sense) (SEQ ID NO: 585) andCATTTAGGATGTGGAGATGAGC (antisense) (SEQ ID NO: 586); Exon 20, GAAACTCAAGATCGCATTCATGC (sense) (SEQ ID NO: 587) and GCAAACTCTTGCTATCCCAGGAG(antisense) (SEQ ID NO: 588); Exon 21, CAGCCATAAGTCCTCGACGTGG (sense)(SEQ ID NO: 589) and CATCCTCCCCT GCATGTGTTAAAC (antisense) (SEQ ID NO:590); Exon 23, GTAGGTTTCTAAACATCAAGAAAC (sense) (SEQ ID NO: 591) andGTGATGACATTTCTCCAGGGATGC (antisense) (SEQ ID NO: 592); Exon 24, CATCACCAATGCCTTCTTTAAGC (sense) (SEQ ID NO: 593) and GCTGGAGGGTTTAATAATGCGATC(antisense) (SEQ ID NO: 594); Exon 25, GCAAACACACAGGCACCTGCTGGC (sense)(SEQ ID NO: 595) and CATTTC CATGTGAGTITCACTAGATGG (antisense) (SEQ IDNO: 596); Exon 26, CACCTTCACAATATACCCTCCATG (sense) (SEQ ID NO: 679) andGACAGCCGTGCAGGGAAAAACC (antisense) (SEQ ID NO: 680); Exon 27,GAACCAGCATCTCAAGGAGATCTC (sense) (SEQ ID NO: 681) andGAGCACCTGGCTTGGACACTGGAG (antisense) (SEQ ID NO: 682).

Nested PCR amplifications for the remaining exons consisted of primaryPCR using the following primers. Exon 1, GACCGGACGACAGGCCACCTCGTC(sense) (SEQ ID NO: 597) and GAAGAACGAAACGTCCCGTTCCTCC (antisense) (SEQID NO: 598); Exon 3, GTTGAGCACT CGTGTGCATTAGG (sense) (SEQ ID NO: 599)and CTCAGTGCACGTGTACTGGGTA (antisense) (SEQ ID NO: 600); Exon 4,GTTCACTGGGCTAATTGCGGGACTCTTGTTCGCAC (sense) (SEQ ID NO: 601) and GGTAAATACATGCTTTTCTAGTGGTCAG (antisense) (SEQ ID NO: 602); Exon 8,GGAGGATGGA GCCTTTCCATCAC (sense) (SEQ ID NO: 603) andGAAGAGGAAGATGTGTTCCTTTGG (antisense) (SEQ ID NO: 604); Exon 9,GAATGAAGGATGATGTGGCAGTGG (sense) (SEQ ID NO: 605) and GTATGTGTGAAGGAGTCACTGAAAC (antisense) (SEQ ID NO: 606); Exon 10, GGTGAGTCACAGGTTCAGTTGC(sense) (SEQ ID NO: 607) and CAAAACATCAGCCATTAACGG (antisense) (SEQ IDNO: 608); Exon 13, GTAGCCAGCATGTC TGTGTCAC (sense) (SEQ ID NO: 609) andCAGAATGCCTGTAAAGCTATAAC (antisense) (SEQ ID NO: 610); Exon 15,CATTTGGCTTTCCCCACTCACAC (sense) (SEQ ID NO: 611) and GACCAAAACACCTTAAGTAACTGACTC (antisense) (SEQ ID NO: 612); Exon 17,GAAGCTACATAGTGTCTCACTTTCC (sense) (SEQ ID NO: 613) andCACAACTGCTAATGGCCCGTTCTCG (antisense) (SEQ ID NO: 614); Exon 22,GAGCAGCCCTGAACTCCGTCAGACTG (sense) (SEQ ID NO: 683) andCTCAGTACAATAGATAGACAGCAATG (antisense) (SEQ ID NO: 684); Exon 28a GCTCCTGCTCCCTGTCATAAGTC (sense) (SEQ ID NO: 615) andGAAGTCCTGCTGGTAGTCAGGGTTG (antisense) (SEQ ID NO: 616); Exon 28b,CTGCAGTGGGCAACCCCGAGTATC (sense) (SEQ ID NO: 617) and CAGTCTGTGGGTCTAAGAGCTAATG (antisense) (SEQ ID NO: 618). Secondary PCRamplification was carried out using primer pairs: Exon 1,GACAGGCCACCTCGTCGGCGTC (sense) (SEQ ID NO: 619) andCAGCTGATCTCAAGGAAACAGG (antisense) (SEQ ID NO: 620); Exon 3, CTCGTGTGCATTA GGGTTCAACTGG (sense) (SEQ ID NO: 621) andCCTTCTCCGAGGTGGAATTGAGTGAC (antisense) (SEQ ID NO: 622); Exon 4,GCTAATTGCGGGACTCTTGTTCGCAC (sense) (SEQ ID NO: 623) and TACATGCTTTTCTAGTGGTCAG (antisense) (SEQ ID NO: 624); Exon 8,CCTTTCCATCACCCCTCAAGAGG (sense) (SEQ ID NO: 625) andGATGTGTTCCTTTGGAGGTGGCATG (antisense) (SEQ ID NO: 626); Exon 9, GATGTGGCAGTGGCGGTTCCGGTG (sense) (SEQ ID NO: 627) and GGAGTCACTGAAACAAACAACAGG(antisense) (SEQ ID NO: 628); Exon 10, GGTTCAGTTGCTTGTATAAAG (sense)(SEQ ID NO: 629) and CCATTAACGGT AAAATTTCAGAAG (antisense) (SEQ ID NO:630); Exon 13, CCAAGGTCATGGAGCACAGG (sense) (SEQ ID NO: 631) andCTGTAAAGCTATAACAACAACCTGG (antisense) (SEQ ID NO: 632); Exon 15,CCACTCACA CACACTAAATATTTTAAG (sense) (SEQ ID NO: 633) andGTAACTGACTCAAATACAAACCAC (antisense) (SEQ ID NO: 634); Exon 17,GAAGCTACATAGTGTCTCACTTTCC (sense) (SEQ ID NO: 635) and CACAACTGCTAATGGCCCGTTCTCG (antisense) (SEQ ID NO: 636); Exon 22,GACGGGTCCTGGGGTGATCTGGCTC (sense) (SEQ ID NO: 685) andCTCAGTACAATAGATAGACAGCAATG (antisense) (SEQ ID NO: 686); Exon 28a,CCTGTCATAAG TCTCCTTGTTGAG (sense) (SEQ ID NO: 637) andGGTAGTCAGGGTTGTCCAGG (antisense) (SEQ ID NO: 638); Exon 28b,CGAGTATCTCAACACTGTCCAGC (sense) (SEQ ID NO: 639) and CTAAGAGCTAATGCGGGCATGGCTG (antisense) (SEQ ID NO: 640). Annealing temperature for exon 1amplifications was 54°. Annealing temperatures for both primary andsecondary amplifications were 58° C. (exons 3, 4, 7-10, 12-17, 19-25,27, and 28), 56° C. (exons 2, 5, 6, and 26), or 52° C. (exons 11 and18).

PCR amplicons were purified using exonuclease I (United StatesBiochemical, Cleveland, Ohio), and shrimp alkaline phosphatase (UnitedStates Biochemical, Cleveland, Ohio) prior to sequencing. Purified DNAwas diluted and cycle-sequenced using the ABI BigDye Terminator kit v1.1(ABI, Foster City, Calif.) according to manufacturer's instructions.Sequencing reactions were electrophoresed on an ABI3100 geneticanalyzer. Electropherograms were analyzed in both sense and antisensedirection for the presence of mutations, using Sequence Navigatorsoftware in combination with Factura to mark heterozygous positions. Allsequence variants were confirmed in multiple independent PCRamplifications and sequencing reactions.

Cancer-Derived Cell Lines:

A panel of 14 lung cancer-derived cell lines was analyzed for EGFRmutations. These were derived from tumors of NSCLC (N=5), small celllung cancer (N=6), adenosquamous (N=1), bronchial carcinoid (N=1), andunknown histology (N=1). Specific cell lines were: NCI-H460, NCI-522,HOP-92, NCIH841, NCIH734, NCIH2228, NCIH596, NCIH727, NCIH446, NCIH1781,NCIH209, NCIH510, NCIH82, NCIH865. In addition, 64 cancer-derived celllines were screened for mutations in exons 19 and 21. These representedthe following histologies: breast cancer (BT549, BT483, UACC893, HS467T,HS578T, MCF7, MCF7-ADR, MDA-MB-15, MDA-MB-175, MDA-MB-231, MDA-MB-415,MDA-MB-436, MDA-MB-453, MDA-MB-468, T47D), ovarian cancer (ES-2,IGROV-1, MDAH2774, OV1063, OVCAR3, OVCAR4, OVCAR5, SKOV3, SW626), CNScancers (SF-295, SNB-19, U-251, CCF-STTG1, SW-1088, SW-1783, T98G,M059K, A172, SK-N-DZ, SK-N-MC), leukemia (CCRF-CEM, K562, MOLT-4,RPMI8226, SR), prostate cancer (DU-145, PC-3), colon cancer (COLO-205,HCT-116, HCT-15, HT-29, SW-620), renal cancer (786-O, ACHN, CAKI-1,SN-12C, UO-31), melanoma (LOX-IMVI, M14, SKMEL2, UACC-62), osteosarcoma(SAOS-2), and head and neck cancers (O11, O13, 019, O28, O22, O29, O12).The head and neck cancer cell-lines were provided by Dr. James Rocco,Massachusetts General Hospital/Massachusetts Eye and Ear Infirmary. Allother cell-lines are available through the American Type CultureCollection (Manassas, Va.).

Genomic DNA was isolated from snap-frozen tumor specimens. Tumorspecimens were first crushed to a fine powder using a pre-chilled andsterilized mortar and pestle. Tumor tissue was immediately transferredinto a DNA extraction solution consisting of 100 mM sodium chloride, 10mM Tris pH7.5, 25 mM EDTA (disodium ethylenediamine tetraacetate) pH8.0,and 0.5% (w/v) sodium dodecyle sulfate, and 100 μg/ml fresh proteinase Kand incubated overnight at 37° C. or for 3 hours at 50° C. DNA was thenextracted using standard phenol-chloroform methods, ethanolprecipitated, washed with 70fi ethanol, air-dried and resuspended in TEbuffer. The DNA concentration was determined spectrophotometrically.Exons 19 and 21 of human EGFR were amplified by the polymerase chainreaction using the following primer pairs: Exon 19 sense primer,5′-GCAATATCAGCCTTAGGTGCGGCTC-3′ (SEQ ID NO: 505); Exon 19 antisenseprimer, 5′-CATAGAA AGTGAACATTTAGGATGTG-3′ (SEQ ID NO: 506); Exon 21sense primer, 5′-CTAACGTTCG CCAGCCATAAGTCC-3′ (SEQ ID NO: 507); Exon21antisense primer, 5′-GCTGCGAGCTCACCCAG AATGTCTGG-3′ (SEQ ID NO: 508).For each sample, 20 ng of genomic DNA was amplified in a PCR reactionconsisting of 1× Expand Long Template buffer 1 (Roche, MannheinGermany), 50 μM sequencing grade dATP (Amersham Biosciences, ClevelandOhio), 50 μM sequencing grade dCTP (Amersham Biosciences, ClevelandOhio), 50 μM sequencing grade dGTP (Amersham Biosciences, ClevelandOhio), 50 μM sequencing grade dTTP (Amersham Biosciences, ClevelandOhio), 0.2 μM sense primer, 0.2 μM antisense primer, 1.25 units ExpandLong Template enzyme mix (Taq DNA polymerase/Tgo DNA polymerase) (Roche,Mannhein Germany) that has been preincubated for 5 minutes on ice with1/6 volume of TaqStart Antibody (1.1 μg/μl) (Clontech, Palo Alto,Calif.) and water to final volume of 25 μl. Each series ofamplifications also includes a negative control for which the DNAtemplate is omitted. PCR cycling conditions for both exons were 95° C.for 2 min followed by 40 cycles of 95° C. for 30 s, 58° C. for 30 s and72° C. for 45 sec; and a final extension of 72° C. for 10 min followedby holding at 4° C. on an MJ-Research PTC-200 or PTC-225 thermal-cycler(MJ-Research, Waltham Mass.).

PCR products were resolved by electrophoresis through a 0.8% agarose gelto ensure amplification from patient material and no amplification inthe negative control. PCR products were purified prior to sequencing bymixing 10 μl each PCR amplicon with 0.5 μl exonuclease I (10 U/μl)(United States Biochemical, Cleveland, Ohio), and 1 μd shrimp alkalinephosphatase (1 U/μl) (United States Biochemical, Cleveland, Ohio) andincubating at 37° C. for 20 minutes followed by inactivation at 80° C.for 15 minutes on a thermal-cycler (MJ-Research, Waltham, Mass.).Purified DNA was diluted in water, according to the intensity of theamplicon, and cycle-sequencing was performed using the ABI BigDyeTerminator kit v1.1 (Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Cycle-sequencing was performed on anMJ-Research thermal-cycler using the following cycling conditions:Primers used for sequencing were: Exon 19 sense primer,5′-GCAATATCAGCCTTAGGTGCGGCTC-3′ (SEQ ID NO: 505); Exon 19 antisenseprimer, 5′-CATAG AAAGTGAACATTTAGGATGTG-3′ (SEQ ID NO: 506); Exon21 senseprimer, 5′-CTAACGTTCGCCAG CCATAAGTCC-3′ (SEQ ID NO: 507) or5′-CGTGGAGAGGCTCAGAGCCTGGCATG-3′ (SEQ ID NO: 687); Exon 21 antisenseprimer, 5′-GCTGCGAGCTCACCCAGAATGTCTGG-3′ (SEQ ID NO: 508). Sequencingreactions were electrophoresed on an ABI3100 genetic analyzer (AppliedBiosystems, Foster City, Calif.). Factura and Sequence Navigator(Applied Biosystems, Foster City, Calif.) software programs were used tomark potential heterozygous positions and display them for evaluation.Nucleotide positions at which the height of the secondary peak wasgreater than, or equal to, 30% the height of the primary peak weremarked as heterozygous and were confirmed by analysis of both sense andantisense reads. Samples with sequence indicative of the presence of amutation were re-amplified and sequenced for confirmation.

-   Position of Primers Used in Sequence Analysis Relative to Exons 19    and 21-   Intronic primers are shown in lower case and underlined.-   Intronic Sequence is Shown in Lowercase.-   Exonic Sequence is Shown in Uppercase.

EGFR Exon 19 (5′-3′) (SEQ ID NO: 641)gcaatatcagccttaggtgcggctccacagccccagtgtccctcaccttcggggtgcatcgctggtaacatccacccagatcactgggcagcatgtggcaccatctcacaattgccagttaacgtcttccttctctctctgtcatagGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATgtgagtttctgctttgctgtgtgggggtccatggctctgaacctcaggcccaccttttctcatgtctggcagctgctctgctctagaccctgctcatctccacatcctaaatgttcactttctatg EGFR Exon 21 (5′-3′) (SEQ ID NO: 642) or(SEQ ID NO: 687) ctaacgttcgccagccataagtcctcgacgtggagaggctcagagcctggcatgaacatgaccctgaattcggatgcagagcttcttcccatgatgatctgtccctcacagcagggtcttctctgtttcagGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAgtaaggaggtggctttaggtcagccagcattttcctgacaccagggaccaggctgccttcccactagctgtattgtttaacacatgcaggggaggatgctctccagacattct gggtgagctcgcagc

Results

Clinical Characteristics of Gefitinib Responders

Patients with advanced, chemotherapy-refractory NSCLC have been treatedwith single agent Gefitinib since 2000 at Massachusetts GeneralHospital. A total of 275 patients were treated, both prior to itsapproval on May 2003 by the FDA, as part of a compassionate use expandedaccess program, and following that date using commercial supply. Duringthis period, 25 patients were identified by clinicians as havingsignificant clinical responses. A significant clinical response wasdefined either as a partial response using RECIST criteria for patientswith measurable disease, or for patients whose tumor burden could not bequantified using these criteria, an evaluable response was assessed bytwo physicians. Table 1 shows clinical characteristics of 9 cases forwhom tumor specimens obtained at the time of initial diagnosis wereavailable. For the other Gefitinib-responders, tissue was not available,most commonly because diagnostic specimens were limited to cytology fromneedle aspirates. As a group, the 9 patients experienced substantialbenefit from Gefitinib. The median survival from the start of drugtreatment is in excess of 18 months, and the median duration of therapyis greater than 16 months. Consistent with previous reports,Gefitinib-responders have a high prevalence of female sex, absence ofsmoking history, and tumors with bronchioalveolar histology (11, 12).Case 6 is representative of the Gefitinib-responsive cohort. Thispatient is a 32 year-old man, without smoking history, who presentedwith multiple brain lesions and disease in the right lung diagnosed asbronchioalveolar carcinoma. He was treated with whole brainradiotherapy, followed by a series of chemotherapy regimens to which histumor did not respond (carboplatin and gemcitabine; docetaxel;vinorelbine). With a declining functional status and progressive lungtumor burden, he started therapy with 250 mg per day of Gefitinib. Hisshortness of breath promptly improved and a lung CT scan 6 weeks afterinitiation of treatment revealed the dramatic improvement shown in FIG.1.

EGFR Mutations in Gefitinib Responders

We hypothesized that cases of NSCLC with striking responses to Gefitinibmight harbor somatic mutations in EGFR, indicating an essential roleplayed by this growth factor signaling pathway in these tumors. Tosearch for such mutations, we first tested for rearrangements within theextracellular domain of EGFR that are characteristic of gliomas (15):none were detected. We therefore sequenced the entire coding region ofthe gene using PCR-amplification of individual exons. Heterozygousmutations were observed in 8/9 cases, all of which were clustered withinthe kinase domain of EGFR (Table 2 and FIG. 2). Four tumors had in-framedeletions removing amino acids 746-750 (delE756-A750; case 1), 747 to750 (delL747-T751insS; case 2), and 747 to 752 (delL747-P753insS; cases3 and 4). The latter two deletions were associated with the insertion ofa serine residue, resulting from the generation of a novel codon at thedeletion breakpoint. Remarkably, these four deletions were overlapping,with the deletion of four amino acids (leucine, arginine, glutamic acidand alanine, at codons 747 to 750) within exon 19 shared by all cases(see FIG. 4 a). Another three tumors had amino acid substitutions withinexon 21: leucine to arginine at codon 858 (L858R; cases 5 and 6), andleucine to glutamine at codon 861 (L861Q; case 7). The L861Q mutation isof particular interest, since the same amino acid change in the mouseegfr gene is responsible for the Dark Skin (dsk5) trait, associated withaltered EGFR signaling (18). A fourth missense mutation in the kinasedomain resulted in a glycine to cysteine substitution at codon 719within exon 18 (G719C; case 8). Matched normal tissue was available forcases 1, 4, 5 and 6, and showed only wild-type sequence, indicating thatthe mutations had arisen somatically, during tumor formation. Nomutations were observed in seven cases of NSCLC that failed to respondto Gefitinib (P=0.0007; 2-sided Fisher's exact test).

Prevalence of Specific EGFR Mutations in NSCLC and Other Cancer Types

Unlike gliomas, in which rearrangements affecting the EGFR extracellulardomain have been extensively studied (15), the frequency of EGFRmutations in NSCLC has not been defined. We therefore sequenced theentire coding region of the gene in 25 primary cases of NSCLC unrelatedto the Gefitinib study, including 15 with bronchioalveolar histology,which has been associated with Gefitinib-responsiveness in previousclinical trials (11, 12). Heterozygous mutations were detected in twobronchioalveolar cancers. Both cases had in-frame deletions in thekinase domain identical to those found in Gefitinib responders, namelydeIL747-P753insS and delE746-A750 (Table 2). Given the apparentclustering of EGFR mutations, we sequenced exons 19 and 21 in a total of55 primary tumors and 78 cancer-derived cell lines, representing diversetumor types (see Supplementary Material). No mutations were detected,suggesting that these arise only in a subset of cancers, in which EGFRsignaling may play a critical role in tumorigenesis.

Increase in EGF-Induced Activation and Gefitinib Inhibition of MutantEGFR Proteins

To study the functional properties encoded by these mutations, theL747-S752insS deletion and the L858R missense mutants were expressed incultured cells. Transient transfection of wild-type and mutantconstructs into Cos-7 cells demonstrated equivalent expression levels,indicating that the mutations do not affect protein stability. EGFRactivation was quantified by measuring phosphorylation of thetyrosine¹⁰⁶⁸ residue, commonly used as a marker of receptorautophosphorylation (19). In the absence of serum and associated growthfactors, neither wild-type nor mutant EGFR demonstratedautophosphorylation (FIG. 3 a). However, addition of EGF led to a 2-3fold increase in receptor activation for both the missense and deletionEGFR mutants, compared with the wild-type receptor. Moreover, whereasnormal EGFR activation was downregulated after 15 min, consistent withreceptor internalization, the two mutant receptors demonstratedcontinued activation for up to 3 hrs (FIG. 3 a). Similar results wereobtained with antibodies measuring total EGFR phosphorylation followingaddition of EGF (not shown).

Since 7/8 EGFR kinase mutations reside near the ATP cleft, which istargeted by Gefitinib, we determined whether the mutant proteins havealtered sensitivity to the inhibitor. EGF-induced receptorautophosphorylation was measured in cells pretreated with variableconcentrations of Gefitinib. Remarkably, both mutant receptors displayedincreased sensitivity to inhibition by Gefitinib. Wild-type EGFR had anIC₅₀ of 0.1 μM and showed complete inhibition of autophosphorylation at2 μM Gefitinib, whereas the two mutant proteins had an IC₅₀ of 0.015 μMand abrogation of autophosphorylation at 0.2 μM (FIG. 3 b). Thisdifference in drug sensitivity may be clinically relevant, sincepharmacokinetic studies indicate that daily oral administration of400-600 mg of Gefitinib results in a mean steady-state trough plasmaconcentration of 1.1-1.4 μM, while the currently recommended daily doseof 250 mg leads to a mean trough concentration of 0.4 μM (20).

Example 2

Tumor cells harboring mutations within the kinase domain of the EGFR,and are therefore sensitive to growth inhibition by gefitinib treatment,can undergo “second-site” mutations, also within the kinase domain, thatconfer resistance to gefitinib but are still “activating” in the sensethat they exhibit increased EGFR signaling relative to wild-type EGFR.Such gefitinib-resistant mutants are generated from two sporadic humanNSCLC cell lines namely NCI-1650 and NCI-1975. Each cell line contains aheterozygous mutation with the kinase domain of EGFR, and is, therefore,expected to be sensitive to gefitinib. The EGFR mutation in NCI-1650consists of an in-frame deletion of 15 nucleotides at position 2235-2249(delLE746-A750) within exon 19, while NCI-1975 has a missense mutationwithin exon 21 that substitutes a G for T at nucleotide 2573 (L858R).The L858R mutation in NCI-H1975 has been shown herein to be activatingand to confer increased sensitivity to gefitinib in vitro.

Gefitinib-resistant cell lines, derived from both NCI-1650 and NCI-1975are isolated, following random chemical mutagenesis using EMS (ethylmethanesulfonate) followed by culture in gefitinib-supplemented mediumto select for the outgrowth of resistant clones. Subcultivation ofindividual clones is followed by nucleotide sequence determination ofthe EGFR gene following specific PCR-mediated amplification of genomicDNA corresponding to the EGFR kinase domain.

A variation of this strategy involves the serial passage of these twocell lines in the presence of gradually increasing concentrations ofgefitinib over a course of several weeks or months in order to selectfor the spontaneous acquisition of mutations within the EGFR gene thatconfer resistance to gefitinib. Selected cells (that continue toproliferate at relatively high gefitinib concentration) are isolated ascolonies, and mutations are identified as described above.

Example 3

To determine whether mutation of receptor tyrosine kinases plays acausal role in NSCLC, we searched for somatic genetic alterations in aset of 119 primary NSCLC tumors, consisting of 58 samples from NagoyaCity University Hospital in Japan and 61 from the Brigham and Women'sHospital in Boston, Mass. The tumors included 70 lung adenocarcinomasand 49 other NSCLC tumors from 74 male and 45 female patients, none ofwhom had documented treatment with EGFR kinase inhibitors.

As an initial screen, we amplified and sequenced the exons encoding theactivation loops of 47 of the 58 human receptor tyrosine kinase genes(*) (Table S1) from genomic DNA from a subset of 58 NSCLC samplesincluding 41 lung adenocarcinomas. Three of the tumors, all lungadenocarcinomas, showed heterozygous missense mutations in EGFR notpresent in the DNA from normal lung tissue from the same patients (TableS2; S0361, S0388, S0389). No mutations were detected in amplicons fromother receptor tyrosine kinase genes. All three tumors had the same EGFRmutation, predicted to change leucine (“L”) at position 858 to arginine(“R”) (FIG. 6A; CTG→CGG; “L858R”), wherein all numbering refers to humanEGFR.

We next examined exons 2 through 25 of EGFR in the complete collectionof 119 NSCLC tumors. Exon sequencing of genomic DNA revealed missenseand deletion mutations of EGFR in a total of 16 tumors, all within exons18 through 21 of the kinase domain. All sequence alterations in thisgroup were heterozygous in the tumor DNA; in each case, paired normallung tissue from the same patient showed wild-type sequence, confirmingthat the mutations are somatic in origin. The distribution of nucleotideand protein sequence alterations, and the patient characteristicsassociated with these abnormalities, are summarized in Table S2.

Substitution mutations G719S and L858R were detected in two and threetumors, respectively. The “G719S” mutation changes the glycine (G) atposition 719 to serine (S) (FIG. 6B). These mutations are located in theGXGXXG motif (SEQ ID NO:490) of the nucleotide triphosphate bindingdomain or P-loop and adjacent to the highly conserved DFG motif in theactivation loop (52), respectively. See, e.g., FIG. 7. The mutatedresidues are nearly invariant in all protein kinases and the analogousresidues (G463 and L596) in the B-Raf protein serine-threonine kinaseare somatically mutated in colorectal, ovarian and lung carcinomas (41,53) (FIGS. 6A, 6B).

We also detected multiple deletion mutations clustered in the regionspanning codons 746 to 759 within the kinase domain of EGFR. Ten tumorscarried one of two overlapping 15-nucleotide deletions eliminating EGFRcodons 746 to 750, starting at either nucleotide 2481 or 2482 of SEQ IDNO: 511 (Del-1; FIGS. 6C and 8C; Table S2). EGFR DNA from another tumordisplayed a heterozygous 24-nucleotide gap leading to the deletion ofcodons 752 to 759 (Del-2; FIG. 6C). Representative chromatograms areshown in FIGS. 8A-8F.

The positions of the substitution mutations and the Del-1 deletion inthe three-dimensional structure of the active form of the EGFR kinasedomain (54) are shown in FIG. 7. Note that the sequence alterationscluster around the active site of the kinase, and that the substitutionmutations lie in the activation loop and glycine-rich P-loop, structuralelements known to be important for autoregulation in many proteinkinases (52).

Two additional EGFR mutations in two different tumor types have beenidentified. Namely, we have identified the EGFR mutation G857V in AcuteMyelogenous Leukemia (AML) and the EGFR mutation L883S in a metastaticsarcoma The “G857V” mutation has the glycine (G) at position 857substituted with a valine (V), while the “L883S” mutation has theleucine (L) at position 883 substituted with a serine (S). Thesefindings suggest that mutations in EGFR occur in several tumor typesand, most importantly, that EGFR inhibitors would be efficacious in thetreatment of patients harboring such mutations. This expands the use ofkinase inhibitors such as, e.g., the tyrosine kinase inhibitorsgefitinib (marketed as Iressa™), erlotinib (marketed as Tarceva™), andthe like in treating tumor types other than NSCLC.

The EGFR mutations show a striking correlation with the differentialpatient characteristics described in Japanese and U.S. patientpopulations. As noted above, clinical trials reveal significantvariability in the response to the tyrosine kinase inhibitor gefitinib(Iressa™), with higher responses seen in Japanese patients than in apredominantly European-derived population (27.5% vs. 10.4%, in amulti-institutional phase II trial) (48); and with partial responsesseen more frequently in the U.S. in women, non-smokers, and patientswith adenocarcinomas (49-51). We show that EGFR mutations were morefrequent in adenocarcinomas (15/70 or 21%) than in other NSCLCs (1/49 or2%); more frequent in women (9/45 or 20%) than in men (7/74 or 9%), andmore frequent in the patients from Japan (15/58 or 26%, and 14/41adenocarcinomas or 32%) than in those from the US (1/61 or 2%, and 1/29adenocarcinomas or 3%). The highest fraction of EGFR mutations wasobserved in Japanese women with adenocarcinoma (8/14 or 57%). Notably,the patient characteristics that correlate with the presence of EGFRmutations appear to be those that correlate with clinical response togefitinib treatment.

To investigate whether EGFR mutations might be a determinant ofgefitinib sensitivity, pre-treatment NSCLC samples were obtained from 5patients who responded and 4 patients who progressed during treatmentwith gefitinib, where these patients were identified out of more than125 patients treated at the Dana-Farber Cancer Institute either on anexpanded access program or after regulatory approval of gefitinib (49).Four of the patients had partial radiographic responses (≧50% tumorregression in a CT scan after 2 months of treatment) while the fifthpatient experienced dramatic symptomatic improvement in less than twomonths. All of the patients were from the United States and wereCaucasian.

While sequencing of the kinase domain (exons 18 through 24) revealed nomutations in tumors from the four patients whose tumors progressed ongefitinib, all five tumors from gefitinib-responsive patients harboredEGFR kinase domain mutations. The Chi-squared test revealed thedifference in EGFR mutation frequency between gefitinib responders (5/5)and non-responders (0/4) to be statistically significant with p=0.0027,while the difference between the gefitinib-responders and unselectedU.S. NSCLC patients (5/5 vs. 1/61) was also significant with p<10⁻¹²(*). The EGFR L858R mutation, previously observed in the unselectedtumors, was identified in one gefitinib-sensitive lung adenocarcinoma(FIG. 6A; Table S3A, IR3T). Three gefitinib-sensitive tumors containedheterozygous in-frame deletions (FIG. 6C and Tables S3A and S3B, Del-3in two cases and Del-4 in one) and one contained a homozygous in-framedeletion (FIG. 6C and Tables S3A and S3B, Del-5). Each of thesedeletions was within the codon 746 to 753 region of EGFR where deletionswere also found in unselected tumors. Each of these three deletions isalso associated with an amino acid substitution (Tables S3A-S3C). In allfour samples where matched normal tissue was available, these mutationswere confirmed as somatic.

Example 3A Primer Design

The cDNA sequences of receptor tyrosine kinases were obtained fromGenBank (accession numbers listed in Table S1), and were to the humangenome assembly (http://genome.ucsc.edu) using the BLAT alignment toidentify exon/intron boundaries. External gene specific primer pairswere designed to amplify exon sequences and at least 250 bp of flankingintronic sequence or adjacent exonic sequence on each side using thePrimer3 program (http://frodo.wi.mit.edu/primer3/primer3_code.html). Theresulting predicted amplicons were then used to design internal primersflanking the exon (generally greater than 50 bp from the exon/intronboundary) and containing appended M13 forward or reverse primer tails.These nested primer sets were tested for appropriate amplicon size andhigh-quality sequence from control DNA. Amplicons encompassing exonsencoding the receptor tyrosine kinase activation loop of 47 tyrosinekinases were amplified and sequenced in a set of 58 primary lung cancersamples from Nagoya City University Medical School. In addition,amplicons covering the full length EGFR were also amplified.

Example 3B PCR and Sequencing Methods for Genomic DNA

Tyrosine kinase exons and flanking intronic sequences were amplifiedusing specific primers in a 384-well format nested PCR setup. Each PCRreaction contained 5 ng of DNA, 1× HotStar Buffer, 0.8 mM dNTPs, 1 mMMgCl2, 0.2 U HotStar Enzyme (Qiagen, Valencia, Calif.), and 0.2 μMforward and reverse primers in a 10 μL reaction volume. PCR cyclingparameters were: one cycle of 95° C. for 15 min, 35 cycles of 95° C. for20s, 60° C. for 30s and 72° C. for 1 min, followed by one cycle of 72°C. for 3 min.

The resulting PCR products were purified by solid phase reversibleimmobilization chemistry followed by bi-directional dye-terminatorfluorescent sequencing with universal M13 primers. Sequencing fragmentswere detected via capillary electrophoresis using ABI Prism 3700 DNAAnalyzer (Applied Biosystems, Foster City, Calif.). PCR and sequencingwere performed by Agencourt Bioscience Corporation (Beverly, Mass.).

Example 3B Sequence Analysis and Validation

Forward (F) and reverse (R) chromatograms were analyzed in batch byMutation Surveyor 2.03 (SoftGenetics, State College, Pa.), followed bymanual review. High quality sequence variations found in one or bothdirections were scored as candidate mutations. Exons harboring candidatemutations were reamplified from the original DNA sample and re-sequencedas above.

Example 3C Patients

Lung tumor specimens were obtained from patients with non-small celllung cancer treated at Nagoya City University Hospital and the Brighamand Womens's Hospital (unselected Japanese tumors and gefitinib-treatedU.S. tumors, respectively) and from the Brigham and Women's Hospitalanonymized tumor bank (unselected U.S. samples) under InstitutionalReview Board approved studies. Information on gender, age, and histologywas available for most samples. Patient samples were also obtained frompatients treated on an open-label clinical trial of gefitinib atDana-Farber Cancer Institute (13). Responses to gefitinib were definedusing standard criteria (See, e.g., A. B. Miller, B. Hoogstraten, M.Staquet, A. Winkler, 1981 Cancer 47, 207-14). IRB approval was obtainedfor these studies.

Of the gefitinib-responsive patients, there were two patients who hadbeen previously treated with at least one cycle of chemotherapy, onepatient previously treated with radiation therapy, one patientconcurrently treated with chemotherapy, and one patient who received noother treatment. For gefitinib-insensitive patients, treatment failurewas defined as the appearance of new tumor lesions or the growth ofexisting tumor lesions in a CT scan after 2 months of gefitinibtreatment compared to a baseline CT scan.

Example 3D cDNA Sequencing of Patient Samples

Total RNA is isolated from tissue samples using Trizol™ (Invitrogen,Carlsbad, Calif.) and is purified using an RNeasy™ mini-elute cleanupkit (Qiagen, Valencia, Calif.). cDNA is transcribed from 2 μg of totalRNA with Superscript II Reverse Transcriptase (Invitrogen Lifetechnologies, Carlsbad, Calif.), according to the manufacturer'srecommendations. The cDNA is used as template for subsequent PCRamplifications of EGFR.

The components of the PCR are: 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5mM MgCl2, 0.1 mM each of dATP, dCTP, dGTP, dTTP, 0.2 μM of each primer,and 0.05 units/μl Taq polymerase (Taq Platinum, GIBCO BRL, Gaithersburg,Md.). Amplification of fragment “a” requires addition of 4% DMSO to thereaction. The primer sequences are listed in Table S4. Forward andreverse primers are synthesized with 18 base pairs of an overhanging M13forward and reverse sequences respectively. The thermocycling conditionsare: 94° C., 4 min; followed by 11 cycles, with denaturing step at 94°C. for 20″, extension step at 72° C. for 20″, and with a 20″ annealingstep that decreased 1° C./cycle, from 60° C. at cycle one to 50° C. atcycle 11; cycle 11 was then repeated 25 times. A 6 minute incubation at72° C. followed by a 4° C. soak completes the program.

An aliquot of the PCR reaction is diluted 1:50 with water. The dilutedPCR product is sequenced using an M13 Forward Big Dye Primer kit(Perkin-Elmer/Applied Biosystems, Foster City, Calif.), according to themanufacturer's recommendations. The sequencing products are separated ona fluorescent sequencer (model 3100 from Applied Biosystems, FosterCity, Calif.). Base calls are made by the instrument software, andreviewed by visual inspection. Each sequence is compared to thecorresponding normal sequence using Sequencher 4.1 software-(Gene CodesCorp.).

Example 3E Tumor Types Expressing Mutant EGFR

Two additional mutations in EGFR were found in two different tumortypes. An EGFR mutation that substitutes a glycine (G) for a valine (V)at position 857 (“G857V”) was identified in Acute Myelogenous Leukemia(AML). An EGFR mutation that substitutes a leucine (L) with a serine (S)at position 883 (“L883S”) in a metastatic sarcoma.

Example 3F Cell Lines

The effects of gefitinib on NSCLC cell lines in vitro were examined. Onecell line, H3255, was particularly sensitive to gefitinib, with an IC50of 40 nM. Other cell lines had much higher IC50s. For example, a wildtype cell line H1666 has an IC50 of 2 uM, which is 50 fold higher thanfor the mutant cell line When the EGFR from this cell line wassequenced, it contained the L858R missense mutation, while the othercell lines were wild type for EGFR. Much lower concentrations ofgefitinib were required to turn off EGFR and also AKT and ERKphosphorylation by EGFR as compared to EGFR wild type cells, whichrequired at least 100 times higher concentrations of gefitinib toachieve the same effect. These findings suggest that the mutant receptoris more sensitive to the effects of gefitinib. Also note here,

Example 3G Combination Therapies

Tumor specimens were analyzed from patients with advanced NSCLC treatedon the randomized trial of carboplatin/paclitaxel with or withouterlotinib. The clinical portion of this trial demonstrated equivalentsurvival in the two treatment arms. Tumor specimens were available forsequencing from 228 of the 1076 patients. The preliminary clinicalcharacteristics of these patients is not different from the group as awhole with respect to baseline demographics, response rate, median andoverall survival.

Exons 18-21 of the tyrosine kinase domain were sequenced and 29mutations, for a mutation frequency of 12.7 percent, were identified.

As a whole the patients with EGFR mutations have a better survivalregardless of whether they received treatment with chemotherapy alone orin combination with erlotinib. These differences are statisticallysignificant with a p value of less than 0.001. These findings raise thepossibility the EGFR mutations, in addition to being predictors ofresponse to gefitinib and erlotinib, may also be prognostic for animproved survival.

(*) Note that the frequency of EGFR mutation in the unselected USpatients, 1 of 61, appears to be low when compared to the frequency ofreported gefitinib response at 10.4%. This difference has a modeststatistical significance (p=0.025 by the chi-squared test). Thus thisresult could still be due to chance, could be due to a fraction ofresponders who do not have EGFR mutations, or could be due to failure todetect EGFR mutations experimentally in this tumor collection. If thefrequency of EGFR mutation in gefitinib-responsive US patients (5/5) iscompared to the expected frequency of gefitinib response (10.4%), thechi-squared probability is again less than 10-12.

Example 4 Study Design

We performed a retrospective cohort study of NSCLC patients referred forsomatic EGFR kinase domain sequencing from August 2004 to January 2005at Massachusetts General Hospital (MGH), Dana-Farber Cancer Institute(DFCI), and Brigham and Women's Hospital (BWH). These three institutionscomprise Dana-Farber/Partners CancerCare (DF/PCC), an academic jointventure cancer center that cares for approximately 1,200 lung cancerpatients per year. In August 2004, EGFR kinase domain sequencing wasmade available for clinical use at DF/PCC. Clinicians could select whichpatients to refer for testing, however patients needed to havesufficient and appropriate tumor specimens available. Tumor cells had tocomprise at least 50% of the specimen based on histologic examination byMGH and BWH reference pathologists, and the specimen had to be from aresection, bronchoscopic biopsy, or core needle biopsy of a primary ormetastatic tumor, or a cellblock from pleural fluid. In rare cases, fineneedle aspirate samples were determined adequate. Samples could beeither paraffin-embedded or frozen tissue. Due to the low incidence ofEGFR mutations in squamous cell tumors (62) patients with this diagnosiswere not eligible for testing.

We identified patients undergoing EGFR testing using the EGFR case logmaintained at the Laboratory for Molecular Medicine (LMM), of theHarvard Medical School/Partners HealthCare Center for Genetics andGenomics (CLIA#22D1005307), the diagnostic testing facility where allsequencing was performed and interpreted. We included all patientsreferred for EGFR testing from DF/PCC with a diagnosis of NSCLC duringthe study period.

Patient age, gender, and race were collected from the electronic medicalrecord system. Smoking status, cancer history, EGFR kinase domainsequencing results, and subsequent EGFR-TKI treatment plans weredocumented using structured physician chart review. Specifically, thesmoking status and cancer history were obtained from physician andnursing notes. Former smokers were defined as patients who had quitsmoking at least one year before their diagnosis of lung cancer andnever-smokers were defined as patients who had smoked less than 100cigarettes in their lifetime. Smokers who had quit within a year oftheir diagnosis or who were smoking at the time of diagnosis wereclassified as current smokers. Pack-years of smoking were calculated bymultiplying the number of packs smoked per day by the number of years ofsmoking. Tumor histology and EGFR kinase domain sequencing results wereobtained from pathology reports. All pathology specimens were centrallyreviewed at either MGH or BWH and histology was categorized using theWorld Health Organization (WHO) classification system (63). Subsequenttreatment plans were obtained from physician notes.

Complete data were available for age, gender, tumor histology, and EGFRmutation status. There were missing data for race (12%), tumor stage attime of testing (4%), smoking status (6%), prior treatments (5%), andsubsequent EGFR-TKI treatment plans (11%). This study was approved bythe Institutional Review Board at DF/PCC.

EGFR Gene Sequencing:

Serial sections of either frozen or formalin-fixed, paraffin-embedded(FFPE) tumor tissue were cut and placed on a glass slide. A region oftumor tissue consisting of at least 50% viable tumor cells wasidentified by a pathologist. FFPE samples were extracted with xylene andethanol to remove paraffin. Both FFPE and frozen tissue samples weredigested with proteinase K overnight. Genomic deoxyribonucleic acid(DNA) was extracted from tissue and peripheral whole blood usingstandard procedures. Genomic DNA was extracted from saliva samples usingthe DNA Genotek-Oragene™ saliva kit.

The kinase domain of EGFR (exons 18-24 and flanking intronic regions)was amplified in a set of individual nested polymerase chain reaction(PCR) reactions. The primers used in the nested PCR amplifications aredescribed in Table S1A and B and SEQ ID 1-424 with the addition ofuniversal sequences to the 5′ ends of the primers (5′tgtaaaacgacggccagt) (SEQ ID NO. 645). The PCR products were directlysequenced bi-directionally by dye-terminator sequencing. PCR wasperformed in a 384-well plate in a volume of 15 μL containing 5 nggenomic DNA, 2 mM MgCl₂, 0.75 μl DMSO, 1 M Betaine, 0.2 mM dNTPs, 20pmol primers, 0.2 μl AmpliTaq Gold® (Applied Biosystems), 1× buffer(supplied with AmpliTaq Gold). Thermal cycling conditions were asfollows: 95° C. for 10 minutes; 95° C. for 30 seconds, 60° C. for 30seconds, 72° C. for 1 minute for 30 cycles; and 72° C. for 10 minutes.PCR products were purified with Ampure® Magnetic Beads (Agencourt).

Sequencing products were purified using Cleanseq™ Magnetic Beads(Agencourt) and separated by capillary electrophoresis on an ABI 3730DNA Analyzer (Applied Biosystems). Sequence analysis was performed byMutation Surveyor (SoftGenetics, State College, Pa.) and manually by tworeviewers. Non-synonymous DNA sequence variants were confirmed byanalysis of 3-5 independent PCR reactions of the original genomic DNAsample. Blood or saliva samples from individuals with non-synonymous DNAsequence variants were analyzed to determine whether the sequencechanges were unique to tumor tissue.

Statistical Analysis:

We constructed logistic regression models to assess the univariateassociation between patient demographic and clinical characteristics andEGFR mutation status. To identify significant predictors of mutationpositive status, we constructed a multivariable logistic regressionmodel including independent variables identified in prior studies aspredictive of mutations, specifically gender, race, histology, andsmoking status. Six patients were excluded from these analyses due tomissing EGFR mutation data as a result of PCR failure. All analyses wereperformed using SAS statistical software (version 8.02, SAS Institute,Cary, N.C.).

Results

Patient Characteristics:

Among the 100 patients with NSCLC referred for somatic EGFR kinasedomain sequencing as part of clinical cancer care during the studyperiod, the mean age was 60.7 years and 63% were female (Table 4). Themajority of patients were white (76%) or Asian (7%), and had metastaticdisease at the time the test was ordered (67%). Nearly all patients(94%) tested for EGFR mutations had adenocarcinoma, adenocarcinoma withbronchioloalveolar carcinoma (BAC) features, or pure BAC. Approximatelyone third of the patients were never-smokers. Therapy administered priorto the referral for EGFR testing included surgery (50%), chestradiotherapy (22%), chemotherapy (47%), and EGFR directed targetedtherapy (11%).

Mutations Identified:

The average length of time from referral for testing to resultavailability was 12 business days. The majority of specimens submittedwere paraffin-embedded (74%). Six of the 74 (8%) paraffin-embeddedspecimens failed PCR amplification, while all of the 26 frozen specimenswere successfully amplified. Among the 94 patients with interpretableresults, 23 (24%) were found to have at least one mutation in the EGFRkinase domain, with two of these patients demonstrating two pointmutations each, for a total of 25 mutations identified (Table 5). Amongthe 23 patients with mutations, 9 (39%) had one or more point mutations,12 (52%) had in-frame overlapping deletions in exon 19 and two patients(9%) had duplications in exon 20. The point mutations were in exons 18and 21, and included five 2818T>G (L858R), and one each of 2371A>T(E709V), 2400G>A (G719S), 2401G>C (G719A), 2573G>A (R776H), 2789C>T(P848L), and 2828T>A (L861Q) of SEQ ID NO: 511 512 respectively. One ofthe point mutations (P848L) was detected in both the tumor specimen andin mononuclear cells obtained from a buccal swab. No mutations weredetected in exons 22, 23, or 24.

Predictors of Mutations:

In our sample, there was no significant association between EGFRmutation status and age (p=0.61), female gender (p=0.92), Asian race(p=0.08), or metastatic disease at the time of referral (p=0.43, Table4). None of the 6 patients with non-adenocarcinoma tumor histology werefound to have mutations. Among the patients with adenocarcinoma,adenocarcinoma with BAC features and pure BAC, there was no associationbetween BAC/BAC features and EGFR mutation status (p=0.35).

None of the 17 current smokers were found to have a mutation.Never-smokers were significantly more likely to have an EGFR mutationthan former smokers (odds ratio [OR]=3.08, 95% confidence interval [CI]1.09-8.76). The mean number of pack-years smoked was significantly loweramong EGFR mutation-positive patients (0.7 pack-years) compared to EGFRmutation-negative patients (25.0 pack-years, p<0.001). For eachadditional pack-year smoked, there was a 4% decrease in the likelihoodof having a mutation (OR=0.96, 95% CI 0.93-0.99).

The number of pack-years of smoking remained a significant predictor ofmutation status after controlling for gender, race, and tumor histology(OR=0.96, 95% CI 0.93-0.99).

Subsequent Use of Test Information:

EGFR mutation-positive patients were significantly more likely to havedocumented plans to receive subsequent EGFR-TKI treatment (86%) thanEGFR mutation-negative patients (11%, p<0.001). Clinicians documentedthat the EGFR results affected their prioritization of recommendedtherapies in 38% of cases. These cases included 14 (61%) of the 23mutation-positive patients for whom EGFR-TKI therapy was recommendedearlier than it would have been had the test been negative, and 24 (34%)of the 71 mutation-negative patients for whom EGFR-TKI therapy was notrecommended, or was recommended later than it would have been had thetest been positive.

EGFR mutation status was more likely to change prioritization oftreatment options in patients with metastatic disease (54%) than inpatients with local or locally advanced disease (19%, p=0.003). Giventhis finding, we further analyzed the decision-making process inmetastatic patients (FIG. 10). Among the 31 patients with metastaticdisease whose test results affected treatment recommendations, fivemutation-positive patients were offered first-line EGFR-TKI treatmentand six mutation-positive patients were offered second-line EGFR-TKItreatment in lieu of chemotherapy. Twenty mutation-negative patientswere encouraged to defer EGFR-TKI treatment until third-line treatmentor beyond based on their negative EGFR test results. Among the 26patients with metastatic disease whose test results did not affecttreatment recommendations, two mutation-negative patients receivedfirst-line EGFR-TKI treatment despite their negative results, ninepatients including four mutation-positive patients received second orthird-line EGFR-TKI treatment, and 15 patients including twomutation-positive patients did not receive a recommendation for anEGFR-TKI. Three of the patients with metastatic disease wereparticipating in trials evaluating first-line EGFR-TKI therapy. Nine ofthe patients with metastatic disease had previously received or werereceiving EGFR-TKIs at the time of EGFR testing.

Discussion

We studied the first 100 patients with NSCLC to undergo screening forsomatic EGFR mutations as part of clinical cancer care at ourinstitution and found that testing was feasible and significantlyimpacted the treatment of NSCLC patients. Patients harboring EGFRmutations were significantly more likely to receive recommendations forEGFR-TKI therapy than patients without mutations. Physicians adjustedtheir treatment recommendations based on the test results in overone-third of the cases, and were more likely to do so in patients withmetastatic disease. In our patient sample, physicians used positive EGFRtest results to help make the decision to prioritize EGFR-TKIs overchemotherapy for some patients, especially for first or second-linetreatment. However, negative EGFR test results did not preventphysicians from administering EGFR-TKIs to selected patients. Many ofthe patients in whom the test result did not impact clinicaldecision-making had early stage, resected disease or were alreadyreceiving an EGFR-TKI for metastatic disease at the time of testing.This is reasonable since the utility of EGFR-TKIs as adjuvant therapy isnot known and there is a benefit to EGFR-TKI therapy in a small numberof patients without an identified EGFR mutation (65, 66-70, 71).

Our study also provides evidence that molecular diagnostics can enhancethe clinical ability to identify patients with EGFR mutations. Manyoncologists currently use the clinical characteristics associated withEGFR mutations and response to EGFR-TKIs to guide the decision-makingprocess for patients with NSCLC. Indeed, our population of patientsreferred for EGFR testing demonstrated an increased prevalence of suchcharacteristics. For example, 95% of referred patients hadadenocarcinoma or BAC tumor histology, compared to 45% in the generalNSCLC population (72). While never-smokers comprised 29% of ourpopulation, the incidence of never-smokers in the general NSCLCpopulation has been reported as 2-10%, and may be as high as 27% inwomen with NSCLC (73-75). Similarly, our population consisted of only17% current smokers, compared to the 38-75% rate of current smokingamong newly diagnosed NSCLC patients (75, 78-80). Our clinicallyselected population consequently had an EGFR mutation rate of 24%, whichis substantially higher than rates documented by our and other U.S.groups that tested unselected available NSCLC tumor samples (65-66, 81).However, it is important to note that while clinicians appeared to beattempting to select patients for testing that had the clinicalcharacteristics predictive of EGFR mutations, the mutation frequency wasstill only 24%, highlighting the fact that molecular diagnosticsincrease the information available to make clinical decisions.

Smoking status was the strongest predictor of EGFR mutation status inour patients, with an increase in smoking history associated with asignificantly decreased likelihood of harboring an EGFR mutation, aftercontrolling for previously described predictors of mutation status. Ourresults are consistent with other case series documenting the importanceof smoking status in the likelihood of EGFR mutations (66, 69, 70, 81,82). Just as the extremely low prevalence of EGFR mutations in squamouscell tumors (62) has shifted testing efforts towards adenocarcinomatumors, it may be appropriate to focus future efforts on patients with alow or absent smoking history. However, reports of EGFR mutations inpatients without typical clinical characteristics advise against stricttesting limitations (83). When examining the other clinicalcharacteristics thought to be associated with mutations, we found Asianrace and BAC tumor histology to have non-significant trends towardspredicting EGFR mutation status. The lack of statistical significance inthese associations may be due to small sample size.

The test was feasible and fit into the time constraints of clinicalcancer care. Nearly all of the tumors submitted for analysis producedinterpretable results. The six specimens that failed PCR amplificationwere all paraffin-embedded, while none of the frozen specimens failedPCR amplification. When available, fresh frozen tissue is the preferablesubstrate for EGFR mutation testing.

There have been close to 2,500 NSCLC samples reported thus far that haveundergone partial or complete EGFR sequence analysis. Our patientsdemonstrated mutations similar to previous reports, with overlappingexon 19 deletions of 9-23 base pairs and point mutations leading tosingle amino acid substitutions in exons 18 and 21. Five of the pointmutations we found have been described above (E709V, G719S, G719A,L858R, and L861Q). One of the point mutations we found causes an aminoacid substitution at a codon where a different amino acid substitutionhas been previously described (R776H). The E709V and R776H variants wereeach found in combination with a known gefitinib-sensitizing mutationinvolving codon 719. The P848L mutation in exon 21 was found in both thesomatic and buccal samples, suggesting it may be a germline variant ofuncertain significance. The patient was a never-smoking female withadenocarcinoma who had stable disease for 15 months on gefitinibtreatment, prior to the EGFR mutation testing. When the P848L mutationwas revealed, she had recently been found to have progressive diseaseand was started on erlotinib therapy. No information about response toerlotinib is available at this time.

The (2499-2522 del of SEQ ID NO: 511) deletion overlaps previouslydescribed exon 19 deletions. The deletions in our patients can becategorized into one of two groups: those spanning codons 747-749 at aminimum (amino acid sequence LRE), and those spanning codons 752-759(FIG. 11). Analysis of all exon 19 deletions reported to date suggeststhat a wide variety of amino acids can be deleted from the TK regionspanning codons 747-759. There does not appear to be a required commoncodon deleted; however, all of the deletions we detected maintained alysine residue at position 745.

One of our two exon 20 mutations are in a never-smoking female withrecurrent adenocarcinoma who was treated with erlotinib after EGFRtesting was performed and has had stable disease for two months at thistime. The other is a former-smoking male with metastatic adenocarcinomawho was treated with an EGFR-TKI, but could not tolerate it due tosevere rash. The identification of clinically relevant EGFR mutations inexon 20 underscores the importance of comprehensive sequencing of the TKregion of EGFR.

In conclusion, this study demonstrates the feasibility and utility ofcomprehensive screening of the TK region of the EGFR gene for somaticmutations in NSCLC patients as part of clinical cancer care. The resultof the test provides useful information regarding clinical predictors ofEGFR-TKI response. Current smokers are less likely to harbor a mutation,as are former smokers with a high number of pack-years of smokinghistory.

Example 5 EGFR Gene Test for Non-Small Cell Lung Cancer, a StandardOperating Procedure

Clinical Indications:

This test is indicated for individuals with Non-Small Cell Lung Cancer.

Analytical Principle

The EGFR Gene Test is a genetic test that detects mutations in thekinase domain of EGFR. DNA is first obtained from a tumor biopsy. TheDNA sequence of 7 exons (18, 19, 20, 21, 22, 23, 24) of EGFR is thendetermined by direct bi-directional gene sequencing. The sequenceobtained is then compared to known EGFR sequence to identify DNAsequence changes. If a DNA sequence change is detected in tumor tissue,the test will be repeated on the original tissue sample. If the changehas not previously been reported in a gefitinib- or erlotinib-responder,the test will also be conducted with a sample of the individual's bloodto determine whether the mutation is constitutive (and therefore likelya normally occurring polymorphism) or occurred somatically in the tumortissue.

Specimen Requirements:

A minimum of 100 ng of DNA is required from tissue sample. Note:Extremely small quantities of DNA may be extracted from tissue samples.The concentration of this DNA may not be accurately quantitated.

Quality Control:

Controls Used

Two negative controls (water) and a positive control (human DNA) foreach exon are included in the PCR reactions. The negative control shouldproceed through the entire procedure to ensure that the sequenceobtained is not the result of contamination. A pGEM positive control andan ABI array control are included in the sequencing step.

Control Preparation and Storage:

The positive control for PCR is either Clontech human DNA or human DNAfrom an anonymous blood sample and is stored at 4° C. The negativecontrol for the PCR reaction is HyPure Molecular Biology Grade waterstored at room temperature. The pGEM positive sequencing reactioncontrol and the ABI array control are stored at −20° C.

Tolerance Limits and Steps to Take if Individual Control Fails:

If the positive PCR control fails but the negative controls and samplespass, the PCR results will be designated as pass and sequencing will beperformed. If a negative control shows evidence of DNA amplification,the whole reaction will be repeated with a new aliquot of patient's DNA.If the pGEM control fails and the test reactions fail, the sequencingrun will be repeated with a second aliquot of the PCR product. If thesequencing controls fail but the test reactions pass, the sequencingdoes not need to be repeated. NOTE: Due to the low yield of DNAextraction from paraffin embedded tissue samples, external PCR reactionsoften do not yield visible products. Internal PCR reactions should yieldvisible products. The size of the product detected on the gel should becompared to the anticipated sizes (see below) to ensure that theappropriate PCR product has been obtained. If an internal PCR product isnot visible on the gel, exon-specific PCR failures should be repeated.

If PCR amplification for an individual sample fails, a new round of PCRshould be attempted with a two-fold increase in input DNA template. IfPCR amplification fails again, a new DNA sample for that patient shouldbe acquired if available. If the sample was a paraffin-embedded tissuesample, additional slides should be scraped. If available, more slidesthan used to generate the original sample should be scraped anddigestion in Proteinase K should be allowed to occur for three nights.

Equipment and Reagents (All reagents stable for one year unlessotherwise noted.)

PCR and Sequencing (in general, PCR and sequencing equipment andreagents are known to those of skill in the art and may be used herein,also noted above).

Primers: (see Table 6 and 7 Below)

TABLE 6 External PCR Primers: Forward  SEQ ID Reverse  SEQ ID ExonPrimer Sequence, (5′→3′) NOS Primer Sequence, (5′→3′) NOS 18TCAGAGCCTGTGTTTCTACCAA 653 TGGTCTCACAGGACCACTGATT 646 19AAATAATCAGTGTGATTCGTGGAG 654 GAGGCCAGTGCTGTCTCTAAGG 647 20ACTTCACAGCCCTGCGTAAAC 655 ATGGGACAGGCACTGATTTGT 648 21GCAGCGGGTTACATCTTCTTTC 656 CAGCTCTGGCTCACACTACCAG 649 22CCTGAACTCCGTCAGACTGAAA 657 GCAGCTGGACTCGATTTCCT 650 23CCTTACAGCAATCCTGTGAAACA 658 TGCCCAATGAGTCAAGAAGTGT 651 24ATGTACAGTGCTGGCATGGTCT 659 CACTCACGGATGCTGCTTAGTT 652

TABLE 7 Internal PCR Primers: Product Length ExonForward Primer Sequence, (5′→3′) Reverse Primer Sequence, (5′→3′) (bp)18 TCCAAATGAGCTGGCAAGTG TCCCAAACACTCAGTGAAACAAA 397 (SEQ ID NO 660)(SEQ ID NO 667) 19 GTGCATCGCTGGTAACATCC TGTGGAGATGAGCAGGGTCT 297(SEQ ID NO 661) (SEQ ID NO 668) 20 ATCGCATTCATGCGTCTTCAATCCCCATGGCAAACTCTTG 378 (SEQ ID NO 662) (SEQ ID NO 669) 21GCTCAGAGCCTGGCATGAA CATCCTCCCCTGCATGTGT 348 (SEQ ID NO 663)(SEQ ID NO 670) 22 TGGCTCGTCTGTGTGTGTCA CGAAAGAAAATACTTGCATGTCAGA 287(SEQ ID NO 664) (SEQ ID NO 671) 23 TGAAGCAAATTGCCCAAGACTGACATTTCTCCAGGGATGC 383 (SEQ ID NO 665) (SEQ ID NO 672) 24AAGTGTCGCATCACCAATGC ATGCGATCTGGGACACAGG 302 (SEQ ID NO 666)(SEQ ID NO 673) F tgtaaaacgacggccagt 5′ end of all forward primers 18primer (SEQ ID NO 645) linker R aacagctatgaccatg 5′end of all reverse primers 16 primer (SEQ ID NO 674) linker

Precautions

TABLE 8 Task Instruction(s) Risk 1. PCR Setup Use PCR Hood Contaminationof PCR Use dedicated pipets reaction and filtered tips Only openreagents in the hood 2. Use of PCR Do not use any post- Contamination ofPCR Hood PCR samples or reaction reagents in the hood

Preparing PCR Reaction Mix for External PCR

All procedures performed in PCR hood for genomic DNA, not the cleanhood.

-   1. Thaw out Taq Gold and dNTP on ice.-   2. Prepare the master mix in a tube (eppendorf or 15 mL tubes) using    the table below. Water, Betaine, 10× Buffer, MgCl₂, DMSO, Taq Gold    and dNTP should be added in the order listed. It is very important    to mix the reagents by pipetting up-and-down gently while adding    each reagent.-   3. DNA should be added to the master mix before aliquoting. After    making the large volume of master mix, aliquot 96 ul (enough for 8    rxns) to a separate tube for each patient or control. Add 8 ul of    DNA at 5 ng/ul to the 96 ul of mastermix. 13 ul can then be added to    the individual wells of the plate or put in strip tubes and pipetted    with a multi-channel pipettor.-   4. For a full 384-well plate of reactions, make enough master mix    for about 415 reactions.-   5. Spin the plate of master mix to get rid of air bubbles.-   6. If using a large set of primers, it would help to have them in    96-well plates with forward primers and reverse primers in separate    plates.-   7. Add the primers using a multi-channel pipette. Make sure to mix    by pipetting up-and-down gently.-   8. Spin the plate to get rid of any air bubbles.-   9. Use the cycle below to amplify.

TABLE 9 Reagent Volume per reaction (μL) Autoclaved ddH₂O 4.90 5MBetaine 3.00 10× Buffer 1.50 Magnesium Chloride 1.50 DMSO 0.75 Taq 0.20dNTP 0.15 PCR Forward Primer1 (conc. 20 pmol/uL) 1.00 PCR ReversePrimer2 (conc. 20 pmol/uL) 1.00 DNA (conc. 5 ng/uL) 1.00 Total volume ofPCR reaction 15.00 Note: PCR is done in 384-well plates.

TABLE 10 PCR Amplification Cycle Activate Taq Gold 10 minutes 95° C.Denature 30 seconds 95° C. 30 cycles Anneal 30 seconds 60° C.  1 minutes72° C. Extend 10 minutes 72° C. Hold ∞  4° C. Note: A cleanup is notnecessary after performing the external PCR.

Preparing PCR Reaction Mix for Internal PCR

The internal PCR set up is almost the same as the external PCR with afew exceptions.

1. Make the large volume of master mix as described for external PCR inthe PCR hood.

2. Aliquot MM to 7 strip tubes and multichannel pipette 12 ul into the384-well plate.

3. Add 1 ul each of forward and reverse internal primers. Temporarilyseal plate.

4. Remove from hood, spin down plate and proceed to post PCR set-uparea.

5. Use dedicated pipettes to aliquot 1 ul of external PCR product intoeach reaction.

6. Heat seal and spin again.

7. Run same amplification cycle as external.

Run PCR products on a 1% gel before clean-up. Determine Pass/Failedexons for repeat PCR.

Clean-Up Internal PCR Using Ampure Magnetic Bead Clean-Up

Cleanup

1. Vortex the plate of Ampure magnetic beads till there is no deposit ofbeads.

2. It is very important that the temperature of the Ampure beads is atroom temperature.

3. Use the 384-well Ampure protocol on the Biomek and change the volumeof reaction to 12 uL to accommodate reagents used for cleanup. If thisis not done, an error will be generated.

4. After the program is complete, hydrate plate with 20 uL of autoclavedddH₂O per well. While adding water, make sure to mix by pipettingup-and-down gently.

5. Spin the plate to get rid of any air bubbles.

6. Place the plate on a magnet to separate out the beads. Now you shouldbe able to take up 1 uL of the DNA to setup sequencing reactions. Savethe rest at −20° C. for future use.

Sequencing Protocol

Preparing Sequencing Reaction Mix

-   -   1. Thaw out BigDye 3.1 in a dark place, on ice.    -   2. Prepare the master mix in a tube (eppendorf or 15 mL tubes)        using the table below. Water, buffer, DMSO and BigDye should be        added in the order listed.    -   3. It is very important to mix the reagents by pipetting        up-and-down gently while adding each reagent.    -   4. When using a universal primer for sequencing, the primer can        also be added to the master mix at this time. If the primer is        unique it should be added individually after the master mix is        in the 384-well plate.    -   5. Usually for a full 384-well plate of reactions, make enough        master mix for about 415 reactions.    -   6. Once the master mix is setup divide the mix into 8 wells of        strip tubes. (Do not use reservoirs to aliquot master mix. That        would be a waste of reagents.) 7.    -   7. Now a multi-channel pipette can be used to aliquot the master        mix into the 384-well plate    -   8. Spin the plate of master mix to get rid of air bubbles.    -   9. Add the PCR product to be sequenced, using a multi-channel        pipette. Make sure to mix by pipetting up-and-down.    -   10. Spin the plate to get rid of any air bubbles.    -   11. Use the cycle below to amplify.

TABLE 11 Reagent Volume per reaction (μL) Autoclaved ddH₂O 4.38 5× ABIBuffer 3.65 DMSO 0.50 ABI BigDye 3.1 0.35 Sequencing Primer 0.12concentration DNA from Internal PCR 1.00 reaction Total Volume ofreaction 10.00

TABLE 12 Amplification Cycle for Sequencing Denature 10 seconds 96° C.25 cycles Anneal  5 seconds 50° C. Extend  4 minutes 60° C. Hold ∞  4°C.

Clean-Up Via Cleanseq Magnetic Bead Clean-Up

-   -   1. Vortex the plate of Cleanseq magnetic beads till there is no        deposit of beads.    -   2. Use the Cleanseq 384-well plate program on the Biomek to        clean-up the samples.    -   3. Once the program is done, save the original plate at −20° C.        The new plate with the clean samples is ready to go on the ABI        3730.

(Note: If the PCR products are shorter than 300 bps you might have todilute the sample before putting it on the 3730)

Create Mutation Surveyor templates for the EGFR test and save them onLMM/Sequencing/Sequences-MS Review/EGFR.

Repeat Result Criteria

All positive results are repeated by amplifying and sequencing thespecific exon(s) in which a DNA sequence change has been detected from asecond aliquot of patient DNA derived from the original tissue sample.In addition, DNA extracted from a sample of the patient's blood shouldbe run in parallel to compare with tumor tissue if the sequence changedetected has not previously been detected in a gefitinib- orerlotinib-responder.

Any exon that did not produce clear sequence will be repeated eitherfrom extraction, PCR or sequencing, based on the specific technicalproblems.

Assay Parameters

Sensitivity of the Test—Somatic EGFR kinase domain mutations have beenfound in approximately 13% of individuals with NSCLC (Paez J G et al.,2004). In addition, somatic EGFR kinase domain mutations have been foundin 13/14 (92.8%) individuals with NSCLC that were gefitinib-responsive(Paez J G et al., 2004, Lynch, et al., 2004). Validation of thetechnical sensitivity of the test demonstrated 100% sensitivity to knownmutations and validation of the sequencing platform in our lab shows100% sensitivity (see “Accuracy of the Technique” below). Thesensitivity for mutation detection of mosaic samples has been determinedto be 25% (i.e., heterozygous mutations can be detected when present at50% of a cell mixture). We have found that up to 20% ofparaffin-embedded tissue do not yield high quality DNA. We are unable toobtain sequence information from these samples.

Specificity of the Test—To date, published literature indicates that noindividuals with a somatic mutation in EGFR were not responsive togefitinib (11/11). The chance of finding a mutation due to an artifactof bi-directional sequencing is close to 0% (see “Accuracy of theTechnique” below). As such, the specificity of the test is approximately100%.

Accuracy of the Technique—The technique of DNA sequencing is the goldstandard in molecular diagnostics. This lab uses the ABI 3730 DNAAnalyzer that has a reported accuracy of 98.5%. Combining this withbi-directional sequencing, automated chromatogram analysis with MutationSurveyor, and manual analysis of false positives, we have achieved anaccuracy rate of 100%. This is based upon an analysis of over 100,000bases of raw sequence. For details of this assessment, see our QualityAssurance Program manual.

Note: We do not assume that these results guarantee 100% accuracy ofthis platform. It is known that sequencing errors can occur and, assuch, we report our accuracy to be 99.99% that has been found by largescale sequencing projects (Hill et al. 2000).

Reproducibility of the Test—Due to the accuracy of the test, whenresults are achieved, they are reproducible equal to the accuracy of thetest (99.99%). However, on occasion, the test can fail due to factorslisted below (see Limitations of Method) or because of PCR or sequencingfailure due to unexplained technical reasons. In these cases, no resultsare achieved and the assay is repeated until a result is achieved or thepatient specimen is deemed unacceptable. Specific rates of failure ofeach assay step and of specimens can be found in the validation reportsin our Quality Assurance Program manual.

Normal Range of the Results—The normal sequence of the EGFR gene can befound online using GenBank accessions: NT_(—)033968.5 (genomic sequence)and NM_(—)005228.3 (mRNA sequence).

Limitations of Method:

Large deletions spanning one or more exons will not be detected by thesequencing method, particularly if present in heterozygosity. Mutationsin the EGFR gene outside of the kinase domain will not be detected bythis assay. Inhibitors may be present in the DNA sample preventingamplification by PCR. Degraded DNA may not produce analyzable data andre-submission of the specimen may be required. Rare sequence variationsor secondary structures of the targeted primer sequences could affectPCR amplification and therefore mutation(s) could be missed in thatregion of one allele.

Example 6

Gefitinib (Iressa) is a tyrosine kinase inhibitor that targets theepidermal growth factor receptor (EGFR), and induces dramatic clinicalresponses in non-small cell lung cancers (NSCLCs) with activatingmutations within the EGFR kinase domain. We report that these mutantEGFRs selectively activate Akt and STAT signaling pathways, whichpromote cell survival, but have no effect on Erk/MAPK signaling, whichinduces proliferation. NSCLCs expressing mutant EGFRs underwentextensive apoptosis following siRNA-mediated knockdown of the mutantEGFR or treatment with pharmacological inhibitors of Akt and STATsignaling, and were relatively resistant to apoptosis induced byconventional chemotherapeutic drugs. Thus, mutant EGFRs selectivelytransduce survival signals on which NSCLCs become dependent;consequently, inhibition of those signals by Gefitinib may underliestriking clinical responses.

Receptor tyrosine kinases of the EGFR family regulate essential cellularfunctions including proliferation, survival, migration, anddifferentiation, and appear to play a central role in the etiology andprogression of solid tumors (R. N. Jorissen et al., Exp. Cell Res. 284,31 (2003), H. S. Earp, T. L. Dawson, X. Li, H. Yu, Breast Cancer Res.Treat. 35, 115 (1995)). EGFR is frequently overexpressed in breast,lung, colon, ovarian, and brain tumors, prompting the development ofspecific pharmacological inhibitors, such as Gefitinib, which disruptsEGFR kinase activity by binding the ATP pocket within the catalyticdomain (A. E. Wakeling et al., Cancer Res. 62, 5749 (2002)). Gefitinibhas induced dramatic clinical responses in approximately 10% of patientswith chemotherapy-refractory NSCLC (J. Baselga et al., J. Clin. Oncol.20, 4292 (2002), M. Fukuoka et al., J. Clin. Oncol. 21, 2237 (2003), G.Giaccone et al., J Clin Oncol. 22, 777 (2004), M. G. Kris et al., JAMA290, 2149 (2003)). Virtually all Gefitinib-responsive lung cancersharbor somatic mutations within the EGFR kinase domain, whereas nomutations have been seen in non-responsive cases (T. J. Lynch et al., N.Engl. J. Med. 350, 2129 (2004), J. G. Paez et al., Science 304, 1497(2004).) These heterozygous mutations include small in-frame deletionsand missense substitutions clustered within the ATP-binding pocket.

Using transient transfections of mutant EGFRs, we showed previously thatboth types of mutations lead to increased EGF-dependent receptoractivation, as measured by autophosphorylation of Y1068, one of theprominent C-terminal phosphorylation sites of EGFR. (T. J. Lynch et al.,N. Engl. J. Med. 350, 2129 (2004).

To enable studies of qualitative differences in signaling by mutantEGFRs, we generated stable lines of non-transformed mouse mammaryepithelial cells (NMuMg) expressing wild-type or mutant EGFRs, andanalyzed EGF-mediated autophosphorylation of multiple tyrosine residueslinked to activation of distinct downstream effectors (R. N. Jorissen etal., Exp. Cell Res. 284, 31 (2003)). Cell lines were generated thatexpressed either wild-type EGFR or one of two recurrent mutationsdetected in tumors from Gefitinib-responsive patients: the missensemutation L858R and the 18 bp in-frame deletion, delL747-P753insS.Significantly different tyrosine phosphorylation patterns were observedbetween wild-type and the two mutant EGFRs at several C-terminal sites.EGF-induced phosphorylation of Y1045 and Y1173 was virtuallyindistinguishable between wild-type and mutant EGFRs, whereasphosphorylation of Y992 and Y1068 was substantially increased in bothmutants. Interestingly, Y845 was highly phosphorylated in the L858Rmissense mutant, but not in the wild-type or the deletion mutant, andhence appears to be unique in distinguishing between the two types ofEGFR mutations. The differential EGF-induced tyrosine phosphorylationpattern seen with wild-type and mutant receptors was reproducible intransiently transfected COS7 cells, ensuring against potential cell typespecific effects.

Thus, Gefitinib-sensitive mutant EGFRs transduce signals that arequalitatively distinct from those mediated by wild-type EGFR. Thesedifferences may result directly from structural alterations within thecatalytic pocket affecting substrate specificity, or from alteredinteractions with accessory proteins that modulate EGFR signaling.

The establishment of cell lines stably transfected with mutant EGFRsmade it possible to compare the phosphorylation status of the majordownstream targets of EGFR in a shared cellular background. EGF-inducedactivation of Erk1 and Erk2, via Ras, of Akt via PLCγ/PI3K, and of STAT3and STAT5 via JAK2, are essential downstream pathways mediatingoncogenic effects of EGFR(R. N. Jorissen et al., Exp. Cell Res. 284, 31(2003)). EGF-induced Erk activation was essentially indistinguishableamong cells expressing wild-type EGFR or either of the two activatingEGFR mutants. In contrast, phosphorylation of both Akt and STAT5 wassubstantially elevated in cells expressing either of the mutant EGFRs.Increased phosphorylation of STAT3 was similarly observed in cellsexpressing mutant EGFRs. The unaltered Erk activation by the mutantEGFRs is consistent with the absence of increased phosphorylation ofY1173, an important docking site for the Shc and Grb-2 adaptors thatleads to Ras activation and subsequent Erk phosphorylation (R. N.Jorissen et al., Exp. Cell Res. 284, 31 (2003)). The increased Akt andSTAT phosphorylation following activation of the mutant EGFRs isconsistent with the increase in Y992 and Y1068 phosphorylation, both ofwhich have been previously linked to Akt and STAT activation (R. N.Jorissen et al., Exp. Cell Res. 284, 31 (2003)). Thus, the selectiveEGF-induced autophosphorylation of C-terminal tyrosine residues withinEGFR mutants is well correlated with the selective activation ofdownstream signaling pathways.

To extend these observations to lung cancer cells in which EGFRmutations appear to drive tumorigenesis, we studied lines derived fromfive NSCL tumors. NCI-H1975 carries the recurrent heterozygous missensemutation L858R and NCI-H11650 has the in-frame deletion delE746-A750,whereas NCI-358, NCI-H1666, and NCI-H1734 express wild-type EGFR. As intransfected cells, EGF-induced autophosphorylation of Y992 and Y1068 wasmarkedly elevated in the two lines with endogenous EGFR mutations, aswas phosphorylation of Akt and STAT5, but not Erk.

The oncogenic activity of EGFR reflects the activation of signals thatpromote both cell proliferation and cell survival (S. Grant, L. Qiao, P.Dent, Front. Biosci. 7, d376 (2002)). While these pathways exhibitoverlap, Ras-mediated activation of the Erk kinases contributessubstantially to the proliferative activity of EGFR, whereas activationof Akt and STATs is largely linked to an anti-apoptotic function (S.Grant, L. Qiao, P. Dent, Front. Biosci. 7, d376 (2002), F. Chang et al.,Leukemia 17, 1263 (2003), F. Chang et al., Leukemia 17, 590 (2003), F.Chang et al., Int. J. Oncol. 22, 469 (2003), V. Calo et al., J. CellPhysiol. 197, 157 (2003), T. J. Ahonen et al., J. Biol. Chem. 278, 27287(2003)). The two lung cancer cell lines harboring EGFR mutationsexhibited a proliferative response to EGF at low serum concentrationsthat was not observed in cells with wild-type receptors. However, theirproliferation rate and cell density at confluence were comparable atnormal serum concentrations.

SiRNA

In contrast, apoptotic pathways were markedly different in lung cancercells with mutant EGFRs: siRNA-mediated specific inactivation of mutantEGFR in these cell lines resulted in rapid and massive apoptosis. About90% of NCI-H1975 cells transfected with L858R-specific siRNA died within96 hours, as did NCI-H1650 cells transfected with delE746A750-specificsiRNA. SiRNA specific for either EGFR mutation had no effect on cellsexpressing the alternative mutation, and siRNA that targets bothwild-type and mutant EGFR had minimal effect on the viability of cellsexpressing only wild-type receptor, but induced rapid cell death inlines expressing EGFR mutants. The ability of siRNAs to specificallytarget the corresponding EGFR alleles was confirmed in transfected COS7cells by immunoblotting. Thus, expression of mutant EGFRs appearsessential for suppression of pro-apoptotic signals in lung cancersharboring these mutations. The fact that lung cancer cells expressingonly wild-type receptors do not display a similar dependence on EGFRexpression may also account for the relative Gefitinib-insensitivity ofhuman tumors that overexpress wild-type EGFR.

The effectiveness of Gefitinib in lung cancers harboring mutant EGFRsmay reflect both its inhibition of critical anti-apoptotic pathways onwhich these cells have become strictly dependent, as well as alteredbiochemical properties of the mutant receptors. We previously reportedthat mutant EGFRs are more sensitive to Gefitinib inhibition ofEGF-dependent autophosphorylation than wild-type receptors (T. J. Lynchet al., N. Engl. J. Med. 350, 2129 (2004)). This increased drugsensitivity by mutant receptors was also observed for both Erk and STAT5activation. Thus, while EGF-induced signaling by mutant receptorsdemonstrates selective activation of downstream effectors viadifferential autophosphorylation events, their enhanced inhibition byGefitinib is uniform, and may reflect altered drug binding to the mutantATP pocket.

To establish the relevance of increased Akt and STAT signaling inEGFR-mediated NSCLC survival, we targeted these pathways with specificpharmacological inhibitors. Lung cancer cells harboring EGFR mutationswere 100-fold more sensitive to Gefitinib than cells with wild-typereceptor. Cells expressing mutant EGFRs were also more sensitive topharmacological inhibition of Akt or STAT signaling than cellsexpressing only wild-type EGFR. While EGFR-mutant lung cancer cellsexhibited increased sensitivity to disruption of Akt/STAT-mediatedanti-apoptotic signals, they demonstrated markedly increased resistanceto cell death signals induced by the commonly used chemotherapeuticagents doxorubicin and cisplatin, and the pro-apoptotic Fas-ligand.

Enhanced Akt/STAT signaling in cells with mutant EGFR might thereforeprovide an additional therapeutic target, while raising the possibilitythat conventional chemotherapy may be less effective against thesetumors.

“Oncogene addiction” has been proposed to explain the apoptosis ofcancer cells following suppression of a proliferative signal on whichthey have become dependent (I. B. Weinstein, Science 297, 63 (2002)).Interestingly, Imatinib (Gleevec) efficiently triggers cell death inchronic myeloid leukemias expressing the BCR-ABL translocation productand in gastrointestinal stromal tumors expressing activating c-Kitmutations, both of which exhibit-frequently constitutive STAT activationthat is effectively inhibited by the drug (T. Kindler et al., Leukemia17, 999 (2003), G. P. Paner et al., Anticancer Res. 23, 2253 (2003)).Similarly, in lung cancer cells with EGFR kinase mutations,Gefitinib-responsiveness may result in large part from its effectiveinhibition of essential anti-apoptotic signals transduced by the mutantreceptor.

Materials and Methods

Immunoblotting

Lysates from cultured cells were prepared in ice-cold RIPA lysissolution (1% Triton X-100, 0.1% SDS, 50 mM Tris-Hcl, pH 7.4, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerol-phosphate, 10 mM NaF, 1 mMNa-orthovanadate, containing protease inhibitors. Debris was removed bycentrifugation in a microfuge at 12,000×g for 10 min at 4° C. Clarifiedlysates were boiled in gel loading buffer and separated by 10% SDS-PAGE.Proteins were electrotransferred to nitrocellulose and detected withspecific antibodies followed by incubation with horseradishperoxidase-conjugated secondary goat antibody (Cell signaling (Beverly,Mass.; 1:2000) and development with enhanced chemiluminescence (DuPontNEN) followed by autoradiography. The phospho-EGFR Y845, Y992, Y1045,Y1068, phospho-STAT5 (tyr694), phospho-AKT (Ser473), phospho-ERK1/2(Thr202/Tyr204), AKT, STAT5, and ERK1/2 antibodies were obtained fromNew England Biolabs (Beverly, Mass.). The total EGFR Ab-20 antibody wasobtained from NeoMarkers (Fremont, Calif.). The phospho-EGFR Y1173antibody was from Upstate Biotechnology (Lake Placid, N.Y.) and thetotal phosphotyrosine antibody PY-20 was from Transduction Laboratories(Lexington, Ky.). All antibodies were used at a 1:1000 dilution.

EGFR Expression Vectors

Full-length EGFR expression constructs encoding the wild type, L858 ordel L747-P753insS mutations were sub-cloned using standard methods intoplasmid pUSEamp. All constructs were confirmed by DNA sequence analysis.

Cell Lines and Transfections

COS7 cells and NMuMg (normal mouse mammary epithelial) cells were grownin DMEM (Dulbecco's modified Eagle's media) with 10% fetal calf serum inthe presence of 2 mM L-glutamine and 50 U/ml penicillin/streptomycin.The NCI-H358, NCI-H1650, NCI-H1734, NCI-H1666, and NCI-H1975 human lungcancer cell lines were obtained from the American Type CultureCollection and were grown in RPMI1640 with 10% fetal bovine serum, 2 mML-glutamine, 50 U/ml penicillin/streptomycin and 1 mM sodium pyruvate.They are referred to in the text, in an abbreviated manner, as H358,H1650, H1734, H1666, and H1975, respectively. Transient transfection ofCOS7 cells was performed using Lipofectamine 2000 (Invitrogen; Carlsbad,Calif.). Plasmid (1 μg) was transfected into cells at 80% confluence ona 10 cm dish. After 12 hours, the cells were harvested and reseeded in12-well plates in the absence of serum. The following day, cells werestimulated with 30 ng/ml of EGF. Stable NMuMg cell lines were preparedby co-transfecting the EGFR expression constructs with thedrug-selectable plasmid pBABE puro, followed by selection in 3 ug/mlpuromycin. Pools of drug-resistant cells were used for analysis.Expression of EGFR in stably transfected cells was confirmed byimmunoblotting.

SiRNA-Mediated “Knockdown” of EGFR Expression

SiRNA for EGFR L858R was designed to target the nucleotide sequenceCACAGATTTTGGGCGGGCCAA (SEQ ID NO.: 688), while the GCTATCAAAACATCTCCGAAA(SEQ ID NO.: 689) sequence was used for the delE745-A750 (Qiagen;Valencia, Calif.). To target all forms of EGFR, commercially preparedsiRNA corresponding to human wild-type EGFR was obtained from Dharmacon(Lafayette, Colo.). Transfection of siRNAs was performed withLipofectamine 2000 (Invitrogen) as per the manufacturer's instructions.Cells were assayed for viability after 96 hours using the MTT assay.

Apoptosis Assay

10,000 cells were seeded into individual wells of a 96-well plate. After6 hours, the medium was changed and the cells were maintained in thepresence of increasing concentrations of doxorubicin (Sigma; St. Louis,Mo.), cisplatin (Sigma), Fas-ligand (human activating, clone CH11;Upstate Biotechnology), Ly294002 (Sigma), or AG490 (Calbiochem; LaJolla, Calif.). After 96 hours, the viability of cells was determinedusing the MTT assay. For caspase immunostaining, 10,000 cells wereseeded onto 10 mm coverslips. The next day they were transfected withsiRNA (see previous section for details). After 72 hours the cells werefixed in 4% paraformaldehyde at room temperature for 10 min. They weresubsequently permeabilized for 5 min in 0.5% Triton X-100 and blockedfor 1 hr in 5% normal goat serum (NGS). The coverslips were thenincubated overnight at 4° C. in primary antibody (cleaved caspase-3Asp175 5A1 from Cell Signaling) at a 1:100 dilution. The next day thecoverslips were washed 3 times in PBS and incubated for 1 hour withsecondary antibody (goat anti-rabbit Texas-red conjugated; from JacksonImmunoresearch; West Grove, Pa.) at a 1:250 dilution in 5% normal goatserum and 0.5 μg/ml of DAPI (4′,6-Diamidino-2-phenylindole). After 3washes in PBS the coverslips were mounted with ProLong Gold anti-fadereagent from Molecular Probes (Eugene, Oreg.).

Cell Viability Assay

10 μl of 5 mg/ml MTT (Thiazolyl blue; Sigma) solution was added to eachwell of a 96-well plate. After 2 hours of incubation at 37° C., themedium was removed and the MTT was solubilized by the addition of 100 μlof acidic isopropanol (0.1N HCL) to each well. The absorbance wasdetermined spectrophotometrically at 570 nm.

Growth Curve

Growth curves for H-358, H-1650, H-1734, and H-1975 cells were obtainedby seeding 1000 cells in individual wells of 96-well plates. Each cellline was plated in 8 separate wells. On consecutive days, the cells werefixed in 4% formaldehyde and stained with 0.1% (w/v) crystal violetsolution. The crystal violet was then solubilized in 100 μl of 10%acetic acid, and the absorbance was measured at 570 nm using a platereader to determine the relative cell number.

Mutation Identification

To identify sporadic NSCLC cell lines harboring mutations within EGFR,we sequenced exons 19 and 21 within a panel of 15 NSCLC cell-lines, asdescribed above. Cell lines were selected for analysis based on theirderivation from tumors of bronchoalveolar histology irrespective ofsmoking history (NCI-H358, NCI-H650, NCI-H1650), or from adenocarcinomasarising within non-smokers (NCI-H1435, NCI-H1563, NCI-H1651, NCI-H1734,NCI-H1793, NCI-H1975, NCI-H2291, NCI-H2342, NCI-H2030, NCI-H1838,NCI-H2347, NCI-H2023). NCI-H1666 has been reported to harbor only wildtype EGFR (see examples above). All cell lines are available from theAmerican Type Culture Collection.

The references cited herein and throughout the specification areincorporated herein by reference in their entirety.

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TABLE 1 Characteristics of Nine Patients with Non-Small-Cell Lung Cancerand a Response to Gefitinib. Age at Beginning Duration of Gefitinib No.of of Over all Patient Therapy Pathological Prior Smoking- TherapySurvival‡ EGFR No. Sex yr Type* Regimens Status^(†) mo Mutation

Response¶ 1 F 70 BAC 3 Never 15.6 18.8 Yes Major; improved lung lesions2 M 66 BAC 0 Never >14.0 >14.0 Yes Major; improved bilater- al lunglesions 3 M 64 Adeno 2 Never 9.6 12.9 Yes Partial; improved lung lesionsand soft- tissue mass 4 F 81 Adeno 1 Former >13.3 >21.4 Yes Minor;improved pleural disease 5 F 45 Adeno 2 Never >14.7 >14.7 Yes Partial;improved liver lesions 6 M 32 BAC 3 Never >7.8 >7.8 Yes Major; improvedlung lesions 7 F 62 Adeno 1 Former >4.3 >4.3 Yes Partial; improved liverand lung lesions 8 F 58 Adeno 1 Former 11.7 17.9 Yes Partial; improvedliver lesions 9 F 42 BAC 2 Never >33.5 >33.5 No Partial; improved lungnodules *Adenocarcinoma (Adeno) with any element of bronchoalveolarcarcinoma (BAC) is listed as BAC. †Smoking status was defined as formerif the patient had not smoked any cigarettes within 12 months beforeentry and never if the patient had smoked less than 100 cigarettes inhis or her lifetime. ‡Overall survival was measured from the beginningof gefitinib treatment to death.

EGFR denotes the epidermal growth factor receptor gene. ¶A partialresponse was evaluated with the use of response evaluation criteria insolid tumors; major and minor responses were evaluated by two physiciansin patients in whom the response could not be measured with the use ofthese criteria.

TABLE 2 Somatic Mutations in the Tyrosine Kinase Domain of EGFR inPatients with Non-Small Cell Lung Cancer Patient Seq. Id. No. MutationEffect of Mutation Patients with a response to gefitinib Patient 1 730Deletion of 15 nucleotides In-frame deletion (746-750) (2235-2249)Patient 2 731 Deletion of 12 nucleotides In-frame deletion (747-751) and(2240-2251) insertion of a serine residue Patient 3 732 Deletion of 18nucleotides In-frame deletion (747-753) and (2240-2257) insertion of aserine residue Patient 4 733 Deletion of 18 nucleotides In-framedeletion (747-753) and (2240-2257) insertion of a serine residue Patient5 734 Substitution of G for T at Amino acid substitution nucleotide 2573(L858R) Patient 6 735 Substitution of G for T at Amino acid substitutionnucleotide 2573 (L858R) Patient 7 736 Substitution of A for T at Aminoacid substitution nucleotide 2582 (L861Q) Patient 8 737 Substitution ofT for G at Amino acid substitution nucleotide 2155 (G719C) Patients withno exposure to gefitinib* Patient A 738 Deletion of 18 nucleotides Inframe deletion (747-753) (2240-2257) and insertion of a serine residuePatient B 739 Deletion of nucleotides In frame deletion (746-750)(2253-2249) *Among the 25 patients with no exposure to gefitinib (15with bronchoalveolar cancer, 7 with adenocarcinoma, and 3 withlarge-cell carcinoma), 2 (Patients A and B) - both of whom hadbronchoalveolar cancer - had EGFR mutations. No mutations were found in14 lung-cancer cell lines representing diverse histologic types:non-small-cell lung cancer (6 specimens), small-cell-lung cancer (6specimens), bronchus carcinoid (3 specimen), and an unknown type (1specimen). Polymorphic variants identified within EGFR included thefollowing: the substitution of A for G at nucleotide 1562, thesubstitution for A for T at nucleotide 1887, and a germ-line variant ofunknown functional significance, the substitution of A for G atnucleotide 2885, within the tyrosine kinase domain.

TABLE 4 Population Characteristics Among 100 Patients Tested for EGFRMutations as Part of NSCLC Care Characteristic Frequency Mean age, years(standard deviation) 60.7 (11.0) Female 63 Race White 76 Asian 7 Other 5Unknown 12 Stage at Time of Test I 15 II 4 III 10 IV 67 Unknown 4Histology Pure BAC 1 Adenocarcinoma with BAC Features 24 Adenocarcinoma69 NSCLC, all other subtypes 6 Smoking Status Current 17 Former 48 Never29 Unknown 6 Mean amount smoked by current and former 39.0 (32.3)smokers, pack-years (standard deviation) Mean time from diagnosis toEGFR test, 18.7 (78.4) months (standard deviation) Prior ChemotherapyTreatment 47 Prior EGFR Targeted Treatment 11 BAC = BronchioloalveolarCarcinoma, EGFR = Epidermal Growth Factor Receptor

TABLE 5 Epidermal Growth Factor Receptor Somatic Gene MutationsIdentified Pack-Years Seq. Patient Gender Histology Smoked ExonNucleotide Change Amino Acid Change Id. No. 1 F Adeno 0 18 2371A > TE709V 740 18 2400G > A G719S 2 F A + BAC 60 18 2401G > C G719A 741 202573G > A R776H 3 F A + BAC 0 19 2481-2495 del K745_A750 del ins K 742 4M A + BAC 0 19 2481-2495 del K745_A750 del ins K 743 5 F Adeno 5 192481-2495 del K745_A750 del ins K 744 6 M Adeno Unknown 19 2481-2495 delK745_A750 del ins K 745 7 F Adeno 0 19 2482-2496 del E746_A750 del 746 8M Adeno 45 19 2482-2496 del E746_A750 del 747 9 F Adeno Unknown 192482-2496 del E746_A750 del 748 10 M A + BAC 12 19 2483-2501 del ins TE746_S752 del ins V 749 11 M Adeno 1 19 2485-2494 del ins C L747_A750del ins P 750 12 M A + BAC 0 19 2485-2497 del ins C L747_T751 del ins P751 13 F Adeno 30 19 2499-2522 del T751_I759 del ins T 752 14 F Adeno 019 2500-2523 del S752_I759 del 753 15 F Adeno 0 20 2549-2557 dupD770_N771 ins SVD 754 16 M Adeno 5 20 2559-2564 dup P772_H773 dup 755CCCCCA 17 F Adeno 0 21 2789C > T P848L* 756 18 M BAC 0 21 2818T > GL858R 757 19 F A + BAC 0 21 2818T > G L858R 758 20 M A + BAC 1 212818T > G L858R 759 21 F Adeno 0 21 2818T > G L858R 760 22 F Adeno 15 212818T > G L858R 761 23 F Adeno 0 21 2827T > A L861Q 762 Adeno =Adenocarcinoma, Adeno + BAC = Adenocarcinoma with BronchioloalveolarCarcinoma Features, BAC = Pure Bronchioloalveolar Carcinoma *Thismutation was identified as a germline variant References to nucleotidenumbers correspond to SEQ ID NO: 511.

TABLE S1A Primers for amplification of selected EGFR and receptortyrosine kinase exons (SEQ ID NOS: 1-212) Gene RefSeq Exon SEQ ID NOF Nested R Nested ALK NM_004304 24 1, 2 GGAAATATAGGGAAGGGAAGGAATTGACAGGGTACCAGGAGATGA ALK NM_004304 25 3, 4 CTGAACCGCCAAGGACTCATTTTTCCCTCCTACTAACACACG AXL NM_021913 19 5, 6 ACTGATGCCCTGACCCTGTTCCCATGGTTCCCCACTCTT CSFIR NM_005211 18 7, 8 AGGGACTCCAAAGCCATGTGCTCTCTGGGGCCATCCACT CSFIR NM_005211 19 9, 10 CATTGTCAAGGGCAATGTAAGTGCTCTCACCAACCCTCGCTGT DDR1 NM_013994 15 11, 12 ACATGGGGAGCCAGAGTGACTGCAACCCAGAGAAAGTGTG DDR2 NM_006182 16 13, 14 TGAGCTTTCAACCCTAGTTTGTTGGTTTGCCTCCTGCTGTCTCA DKFZp761P1010 NM_018423 8 15, 16TGTCCTTGTGTTTTTGAAGATTCC TGACAGACAGATGACAAACATGAA EGFR NM_005228 217, 18 TGGGTGAGTCTCTGTGTGGAG CATTGCCATAGCAAAAATAAACACA EGFR NM_005228 319, 20 GGTTCAACTGGGCGTCCTA CCTTCTCCGAGGTGGAATTG EGFR NM_005228 4 21, 22CGCACCATGGCATCTCTTTA AAAACGATCTCTATGTCCGTGGT EGFR NM_005228 5 23, 24CAGCCAGCCAAACAATCAGA TCTTTGGAGTCTTCAGAGGGAAA EGFR NM_005228 6 25, 26TGTGGTTTCGTTGGAAGCAA AATTGACAGCTCCCCCACAG EGFR NM_005228 7 27, 28GGCTTTCTGACGGGAGTCAA CCACCCAAAGACTCTCCAAGA EGFR NM_005228 8 29, 30CCTTTCCATCACCCCTCAAG AGTGCCTTCCCATTGCCTAA EGFR NM_005228 9 31, 32ACCGGAATTCCTTCCTGCTT CATCTGAAACAAACAACAGGGTGA EGFR NM_005228 10 33, 34AGGGGGTGAGTCACAGGTTC TCAGAAGAAATGTTTTTATTCCAAGG EGFR NM_005228 11 35, 36GCAAATCCAATTTTCCCACTT GCAGGGAGCTCTGTGCCCTAT EGFR NM_005228 12 37, 38TCCCACAGCATGACCTACCA TTTGCTTCTTAAGGAACTGAAAA EGFR NM_005228 13 39, 40TGTCACCCAAGGTCATGGAG CAAAAGCCAAGGGCAAAGAA EGFR NM_005228 14 41, 42GGAGTCCCAACTCCTTGACC GTCCTGCCCACACAGGATG EGFR NM_005228 15 43, 44GCTTTCCCCACTCACACACA CAAACCTCGGCAATTTGTTG EGFR NM_005228 16 45, 46CCACCAATCCAACATCCAGA TGGCCCAGAGCCATAGAAAC EGFR NM_005228 17 47, 48TTCCAAGATCATTCTACAAGATGTCA GCACATTCAGAGATTCTTTCTGC EGFR NM_005228 1849, 50 TCCAAATGAGCTGGCAAGTG TCCCAAACACTCAGTGAAACAAA EGFR NM_005228 1951, 52 GTGCATCGCTGGTAACATCC TGTGGAGATGAGCAGGGTCT EGFR NM_005228 2053, 54 ATCGCATTCATGCGTCTTCA ATCCCCATGGCAAACTCTTG EGFR NM_005228 2155, 56 GCTCAGAGCCTGGCATGAA CATCCTCCCCTGCATGTGT EGFR NM_005228 22 57, 58TGGCTCGTCTGTGTGTGTCA GGAAAGAAAATACTTGCATGTCAGA EGFR NM_005528 23 59, 60TGAAGCAAATTGCCCAAGAC TGACATTTCTCCAGGGATGC EGFR NM_005228 24 61, 62AAGTGTCGCATCACCAATGC ATGCGATCTGGGACACAGG EGFR NM_005228 25 63, 64GGCACCTGCTGGCAATAGAC TGACTTCATATCCATGTGAGTTTCACT EGFR NM_005228 2665, 66 TATACCCTCCATGAGGCACA GGGAAAACCCACACAGGAA EGFR NM_005228 27 67, 68TCAGAACCAGCATCTCAAGGA GATGCTGGAGGGAGCACCT EGFR NM_005228 28_1 69, 70CCTTGTTGAGGACATTCACAGG ATGTGCCCGAGGTGGAAGTA EPHA1 NM_005232 14 71, 72GGAGGGCAGAGGACTAGCTG GTGCCTGGCCAAGTCTTTGT EPHA1 NM_005232 15 73, 74CTGCAGCCTAGCAACAGAGC AAGAACCAGAGGAGCCAGGA EPHA2 NM_004431 13 75, 76CGGGTAAGGATGTGGGTTGT CAGGTGTTCTGCCTCCTGAA EPHA2 NM_004431 14 77, 78GCTTCAGGAGGCAGAACACC GGAGCAAGCCTAAGAAGGTTCA EPHA3 NM_005233 30 79, 80GCCTTGTATCCATTTGCCACA TGACAACACGTTTTGGGTCAT EPHA3 NM_005233 31 81, 82TGCATATTCCATTTCAGAACAGA AAACAGTTTCATTGCTGCTAAAT EPHA4 NM_004438 1383, 84 CCGGATACAGATACCCAAAAAGA GGAGGCTTCAAGGGATGAGA EPHA4 NM_004438 1485, 86 GCTGTTGTCCTGCTTGGCTA TGGTTGTAATGTTGAACTAGCTTGC EPHA7 NM_004440 1387, 88 TGGCTGTCAGCTAAATAAGCATGT TCAATTTGCTTCATTTCTCCTGTT EPHA7 NM_00444014 89, 80 TGCTGCTGAACTACCAACCAA TGTGGTAGTAATTGTGGAAAACTG EPHA8 NM_02052613 91, 92 CAAAGCACCGTCTCAACTCG CCCGAAATGCCAACTTCAT EPHA8 NM_020526 1493, 94 GGAAAACAGGACCCCAGTGT CCCTCCTCCACAGCTGAT EPHB1 NM_004441 7 95, 96GACAGAAGCTGACAAGCAGCA AGGTTCCATTCCCTCCCAGT EPHB1 NM_004441 8 97, 98TGGGAGTGAGAGTTTGGAAGAA TATGAGGCCGTGAGCTGAAA EPHB2 NM_017449 11 99, 100AGGGCCCTGCTCTGGTTT CCAATTGGGCGTTAGTGAAA EPHB2 NM_017449 12 101,CTCATGAGATTGGGGCATCAA AGGCCCATGATCTCAGAAGC EPHB3 NM_004443 11 103,GGTTGCAGGAGAGACGAGGT AGGCCCTTCACCCTGTGAC EPHB3 NM_004443 12 105ATGACCCCTCCGATCCTACC TAATCCTGCTCCACGGCATT EPHB4 NM_004444 14 107GGAAAAAGCAGAGGCAGGTG TGGTCTCAAGAACCCAGCAG EPHB6 NM_004445 16 109GACACCCTCCCCCTCTCAT ACTATGACACCCCGGCTGAG EPHB6 NM_004445 17 111TGCTTGATGTAAAACCCTTGG GCAATCCAACAGCCATGAGA ERBB2 NM_004448 21 113GGAGCAAACCCCTATGTCCA TCCTCCAACTGTGTGTTGTGG ERBB3 NM_001982 21 115TGGGGACCACTGCTGAGAG TGCAGCCTTCTCTCCTTGAA FGFR1 NM_000604 14 117GCAGAGCAGTGTGGCAGAAG ACAGGTGGGAAGGGACTGG FGFR1 NM_000604 15 119AGTGGGGTGGGCTGAGAAC TCTCTGGGGCAGAAAGAGGA FGFR2 NM_000141 14 121ACCCGGCCACACTGTATTTC CATCCCACCCAGCTCTCAAC FGFR2 NM_000141 15 123AGGGCATAGCCCTATTGAGC CCCAGGAAAAAGCCAGAGAA FGFR3 NM_000142 13 125CAGGTGTGGGTGGAGTAGGC CTCAGGCGCCATCCACTT FGFR3 NM_000142 14 127AAGAAGACGACCAACGTGAGC AGGAGCTCCAGGGCACAG FGFR4 NM_002011 14 129CCTCCTCTGTAAAGTGGGTGGA AGAGGGCCTCAGTGCAGAGT FGFR4 NM_002011 15 131AGATGGGGCAGAACTGGATG GGTCCCAGACCAAATCTGA FLT1 NM_002019 23 133AGGTGCTCCCTTCACAGCAT TTCAGGGACTACAGCTGAGGAA FLT1 NM_002019 24 135GCCGTATGTTATCTGGGAGGT TGGGCCCATTACACTTTAAGA FLT3 NM_004119 20 137TTCCATCACCGGTACCTCCT CCATAAATCAAAAATGCACCACA FLT3 NM_004119 21 139GAGTGGTCTTAGGAAGATGATGC AAAGTCATGGGCTGCAATACAA FLT4 NM_002020 23 143ATGGTCCCCACTGCTTGG AGGAGCTCACCTCACCCTGT IGFIR NM_000875 18 143CCTTGCGTCTCTCCACACAT TGGCAACGGGTAACAATGAA INSR NM_000208 18 145GGCTGAGGTAAGCTGCTTCG AAAAAGAAGTATCTTGCCCCTTT INSR NM_000208 19 147AACCCCTCTTAGGGCTCTGTG CAGGAGGATGGCAGGCTTC KDR NM_002253 24 151CGTAGAGAGCTTCAGGACCTGTG TTCCGAGAAGTTTTGCCTGA KIT NM_000222 17 151TGTGAACATCATTCAAGGCGTA AAAATGTGTGATATCCCTAGACAGG KIT NM_000222 18 153TCCATTTCAGCAACAGCA GGCTGCTTCCTGAGACACAGT LTK NM_002344 16 155TATCTACCGGTGCGGGACTT AGGTGTAGCCTCCCCTCACA MERTK NM_006343 17 157AGGCTGGTGGTGTCTCTGTG CAAGCTGCCAACCCTCAGTT MET NM_000245 19 159TGGATTTCAAATACTGAAGCCACT TGGAATTGGTGTTGAATTT MUSK NM_005592 15_1 161GGGCTTCATATGTTCTGACATGG CAGAGGACCACGCCATAGG MUSK NM_005592 15_2 163CCGAGATTTAGCCACCAGGA CCTGGGAAGCAAACAACACA NTRK1 NM_002529 15 165AGGTCCCCAGTCTCCTCTCC AGACCCATGCAGCCATCCTA NTRK1 NM_002529 16 167CGTGAACCACCGAGCTTGT AGAGGGGCAGAAGGGGAAC NTRK2 NM_006180 15 169GGTGGGGGTGAGGAGCTTAG TCGTTTAAGCCACCCAGTTCA NTRK2 NM_006280 16 171TGCAAATAAGGAAAGCAAACA TCCTGACATGGTCTTCCAACC NTRK3 NM_002530 17 173CAGCATCTTCACACACCTCTGA GCTGGCTCTAAATCCCACCT NTRK3 NM_002530 18 175CTAATCCGGGAAGTTGTTGC TTCTGTATCAGCAGCTTCTCTGTG PDGFRA NM_006206 18 177CAAGTGCCACCATGGATCA GGCAGTGTACTGACCCCTTGA PDGFRA NM_006206 19 179GCACAAGTTATTAAGAGCCCAAGG AGCATACTGGCCTCACACCA PDGFRB NM_002609 18 181GCACATGGGCAGTGTTTGTATTT GAGCCCCACACAGATTTCCT PDGFRB NM_002609 19 183ATGGGACGGAGAAGTGGTTG TCCCTGTATCAGGGCTCGTC PTK7 NM_002821 18 185TTCCTACGCAGCACACCAAT GCAGGCACTAAACCCTTTCC PTK7 NM_002821 19 187GCACGCATGTGACCAATTTC AGCCCTGAGAGGGAGGTAGG RET NM_000323 15 189CACACACCACCCCTCTGCT AAAGATTTGGGGTGAGGGCTA RET NM_000323 16 191CTGAAAGCTCAGGGATAGGG CTGGCCAAGCTGCACAGA ROR1 NM_005012 09_1 193TGCAGCCAACGATTTGAAAG GGAAAGCCCCAAGTCTGAAA ROR1 NM_005012 09_2 195TCATCATGAGATCCCCACACT GCATTTCCCCCTGAAGGAGT ROR1 NM_005012 09_3 197TGGATTCAGTAACCAGGAAGTGA CCCATTCCACCAGGATGATT ROR1 NM_005012 09_4 199GTTTCCAGCTGCCCACTACC GCTCGAAACCACATGTTCCA RYK NM_002958 13 201CTGGATTTGGGGTTCTCTGC CGGGAACAGCTAGCAGATTTTT TEK NM_000459 18 203GGGAATTTTGGAGGGGAACT GCTTCAGTCACCACAGAGCA TEK NM_000459 19 205TGAGTCTACCCAGCAATCATTTG TTCCCGAGACTACAGGACA TIE NM_005424 18 207GGTAACAAGGGTACCCACGAA GTTTGAGGGGCTGAGTGTGG TIE NM_005424 19 209CCTCACCCTTAGGGCTTGTG AGCCCAGGTCATGCCTTAGA TYRO3 MM_006293 18 211, 212GGGTAGCTTGGGAGCAAAGA CCAAACCCCAGAGAGCAGAC

TABLE S1B Primers for amplification of selected EGFR andreceptor tyrosine kinase exons (SEQ ID NOS: 213-424) Gene RefSeq ExonSEQ ID NO F External R External ALK NM_004304 24 213, 214CATTTCCCCTAATCCTTTTCCA GTGATCCCAGATTTAGGCCTTC ALK NM_004304 25 215GCCTCTCGTGGTTTGTTTTGTC CCCAGGGTAGGGTCCAATAATC AXL NM_021913 19 217CTTCCTGGTGGAGGTGACTGAT CAGGCATAGTGTGTGATGGTCA CSF1R NM_005211 18 219TCACGATACACATTCTCAGATCC GAAGATCTCCCAGAGGAGGATG CSF1R NM_005211 19 221CGTAACGTGCTGTTGACCAAT AAACGAGGGAAGAGCCAGAAAG DDR1 NM_013994 15 223TGGGGAGCACAATAAAAGAAGA ACTCTTGGCTCCTGGATTCTTG DDR2 NM_006182 16 225GGAAGTCAGTGTGCAGGGAATA TTTTAGCAGAAATAGGCAAGCA DKFZp761P1010 NM_018423 8227 TGGTAATCCTAAACACAATGCAGA CTGGGCAACACAGTGAGATCCT EGFR NM_005228 2 229TCACAAATTTCTTTGCTGTGTCC CATGGAACTCCAGATTAGCCTGT EGFR NM_005228 3 231GATTGTTGCAGATCGTGGACAT CGCTTAAATCTTCCCATTCCAG EGFR NM_005228 4 233CTCCATGGCACCATCATTAACA CTCAGGACACAAGTGCTCTGCT EGFR NM_005228 5 235GCAGTTCATGGTTCATCTTCTTTT CAAAATAGCCCACCCTGGATTA EGFR NM_005228 6 237CTTTCTGCATTGCCCAAGATG CAAGGTCTCAGTGAGTGGTGGA EGFR NM_005228 7 239GAGAAGGGTCTTTCTGACTCTGC CAGGTGTTTCTCCTGTGAGGTG EGFR NM_005228 8 241CACATTGCGGCCTAGAATGTTA ACCCCGTCACAACCTTCAGT EGFR NM_005228 9 243GCCGTAGCCCCAAAGTGTACTA TCAGCTCAAACCTGTGATTTCC EGFR NM_005228 10 245CTCACTCTCCATAAATGCTACGAA GACTTAACGTGTCCCCTTTTGC EGFR NM_005228 11 247GCCTCTTCGGGGTAATCAGATA GAAGTCTGTGGTTTAGCGGACA EGFR NM_005228 12 249ATCTTTTGCCTGGAGGAACTTT CAGGGTAAATTCATCCCATTGA EGFR NM_005228 13 251CAGCAGCCAGCACAACTACTTT TTGGCTAGATGAACCATTGATGA EGFR NM_005228 14 253TGAATGAAGCTCCTGTGTTTACTC ATGTTCATCGCAGGCTAATGTG EGFR NM_005228 15 255AAAACAGGGAGAACTTCTAAGCAA CATGGCAGAGTCATTCCCACT EGFR NM_005228 16 257CAATGCTAGAACAACGCCTGTC TCCCTCCACTGAGGACAAAGTT EGFR NM_005228 17 259GGGAGAGCTTGAGAAAGTTGGA ATTTCCTCGGATGGATGTACCA EGFR NM_005228 18 261TCAGAGCCTGTGTTTCTACCAA TGGTCTCACAGGACCACTGATT EGFR NM_005228 19 263AAATAATCAGTGTGATTCGTGGAG GAGGCCAGTGCTGTCTCTAAGG EGFR NM_005228 20 265ACTTCACAGCCCTGCGTAAAC ATGGGACAGGCACTGATTTGT EGFR NM_005228 21 267GCAGCGGGTTACATCTTCTTTC CAGCTCTGGCTCACACTACCAG EGFR NM_005228 22 269CCTGAACTCCGTCAGACTGAAA GCAGCTGGACTCGATTTCCT EGFR NM_005228 23 271CCTTACAGCAATCCTGTGAAACA TGCCCAATGAGTCAAGAAGTGT EGFR NM_005228 24 273ATGTACAGTGCTGGCATGGTCT CACTCACGGATGCTTGCTTAGTT EGFR NM_005228 25 275TAAGGCACCCACATCATGTCA TGGACCTAAAAGGCTTACAATCAA EGFR NM_005228 26 277GCCTTTTAGGTCCACTATGGAATG CCAGGCGATGCTACTACTGGTC EGFR NM_005228 27 279TCATAGCACACCTCCCTCACTG ACACAACAAAGAGCTTGTGCAG EGFR NM_005228 28_1 281CCATTACTTTGAGAAGGACAGGAA TATTCTTGCTGGATGCGTTTCT EPHA1 NM_005232 14 283AGGAGGGCAGAGGACTAGCTG GGCAATGTGAATGTGCACTG EPHA1 NM_005232 15 285CTTGAACCTGGGAGGTGGAG ATCAGGGTGGGAGGAGTAAAGA EPHA2 NM_004431 13 287CCCACTTACCTCTCACCTGTGC GTGAACTTCCGGTAGGAAATGG EPHA2 NM_004431 14 289AGGGGACCTCAAGGGAGAAG AGATCATGCCAGTGAACTCCAG EPHA3 NM_005233 10 291GGACCAGGAAAGTCCTTGCTTT GGTGGGGAACATTAAACTGAGG EPHA3 NM_005233 11 293GCTTCAGGTTGTTTTGTTGCAG ACCCTTGCTTGAGGGAAATATG EPHA4 NM_004438 13 295CCCAGCTCCTAGGGTACAGTCT CAGTCAGCTTCAAAATCCCTCTT EPHA4 NM_004438 14 297TCACTTCCCTGTGAGTAAAGAAAA GGCCATTTAATTCTTGTCCTTGA EPHA7 NM_004440 13 299TGGACTTGTGCAAACTCAAACTG TCCCAATATAGGGCAGTCATGTT EPHA7 NM_004440 14 301TCTCAATCAGTTGAGTTGCCTTG AGCTGTGCAAGTGTGGAAACAT EPHA8 NM_020526 13 303GCTGTGAGGGTAAATGAGACCA GTCTCCTGGTGAGTGACTGTGG EPHA8 NM_020526 14 305CCTTCCTTCGTCTCCACAGC GTCCTTGTGCCAACAGTCGAG EPHB1 NM_004441 7 307GCTTGGCAAGGAGAAGAGAACA GCTTGCTTTCTTGCTTGAACAAC EPHB1 NM_004441 8 309GCTGGTCACCTTGAGCTTCTCT CCATGCTGGGCTCTTTGATTA EPHB2 NM_017449 11 311CACCACTCTGAAGTTGGCCTCT ATGGCTCTGCACATTTGTTCC EPHB2 NM_017449 12 313CAGAGTGGGAAAAGGCACTTCA CCAGAGTCCTGTGCAGACATTC EPHB3 NM_004443 11 315ATGGGGATTAACTGGGATGTTG CGTAGCTCCAGACATCACTAGCA EPHB3 NM_004443 12 317GCAACCTGGTCTGCAAAGTCTC ACCCAGCAGTCCAGCATGAG EPHB4 NM_004444 14 319GAGTTTCAGTGAGCCAAGATCG TTACAGGCTTGAGCCACTAGGC EPHB6 NM_004445 16 321AAGCTTCCAGGAGACGAGGTC GTCCCTGAAATCCCTCAAACC EPHB6 NM_004445 17 323TGCTCCATAAACGTGACTATTGC GTAAGAGGGTGGGCTGGAATCT ERBB2 NM_004448 21 325CTTAGACCATGTCCGGGAAAAC CACATCACTCTGGTGGGTGAAC ERBB3 NM_001982 21 327AAATTTCATCCCAAAACCAACC CCAGTCCCAAGTTCTTGATCATT FGFR1 NM_000604 14 329ACAAGTCGGCTAGTTGCATGG TCTCAGATGAAACCACCAGCAC FGFR1 NM_000604 15 231TTCATCTGAGAAGCAAGGAGTGG CCAGGGAGAAAGCAGGACTCTA FGFR2 NM_000141 14 233TTCTGGCGGTGTTTTGAAATTA CTCAACATTGACGGGCCTTTCTT FGFR2 NM_000141 15 235TCAGCTCTTAAACAGGGCATAGC GAAATGCAGCAGCCACTAAAGA EGFR3 NM_000142 13 237CTCACCTTCAAGGACCTGGTGT CAGGGAGGGGTAGAAACCACA FGFR3 NM_000142 14 239GGAGAGGTGGAGAGGCTTCAG GAGACTCCCAGGACAGACACCT FGFR4 NM_002011 14 341CACTCGTTCCTCACCCTTCC AGGACTCACACGTCACTCTGGT FGFR4 NM_002011 15 343GGACAATGTGATGAAGATTGCTG ATAGCAGGATCCCAAAAGACCA FLT1 NM_002019 23 345GGCTTGGGGACCTGTATTTGTA CAGTGGCCTTTCTGAGCCTTAC FLT1 NM_002019 24 347GCACTCTAGCTCCCTCTTTTAGC TTTTACAGTAGAGGGCAGACATGC FLT3 NM_004119 20 349GCCACCATAGCTGCAGAATTAG CCCAAGGACAGATGTGATGCTA FLT3 NM_004119 21 351GCCTTTGTTCGAGAGGAGTTGT GTTCACGCTCTCAAGCAGGTTA FLT4 NM_002020 23 353ATTCCACAAGCTCTCTCCATGA CTTGCCCCAAGATGCCTAAG IGFIR NM_000875 18 355TGCTTGGTATTTGCTCATCATGT CCCTTAGCTAGCCCACTGACAA INSR NM_000208 18 357CTCCTGGGAGTGGTGTCCAA CCTGGGCAACAGACAGAGTAAG INSR NM_000208 19 359CTTCACTTCCCCATGCGTACC GGGTTCACAATGCCTACAGGA KDR NM_002253 24 361AAAATCTGTGACTTTGGCTTGG GGGAGGAGACATTCTTTGATTTG KIT NM_000222 17 363GCAGTCCTGAGAAGAAAACAGC CTTCACATGCCCCAAAATTACA KIT NM_000222 18 365TGAGCCATGTATTTCAGAGGTGA TACATTTCAGCAGGTGCGTGTT LTK NM_002344 16 367TTGCCTACTCTGTAGGGATATGC ATAGGGCATGTAGCCCAGTGA MERTK NM_006343 17 369GCTCTGCTGTTGGTCCTCACT TTGCAAAGCACACATCTTCTGA MET NM_000245 19 371TGGCAATGTCAATGTCAAGCAT GTATGTTGCCCCACTCAACAAA MUSK NM_005592 15_1 373TGCATTTCCTAGCTGAGACTCC TGCCATCTCGCACGTAGTAAAT MUSK NM_005592 15_2 375CTCTCCTGTGCTGAGCAGCTTT TGTTTCCAATCACTGGCTTTCA NTRK1 NM_002529 15 377GAACCATGGGCTGTCTCTGG ATCTGGGATAGCGAAGGAGACA NTRK1 NM_002529 16 379ATTACAGGCCACACGCCATC AAGGCAAGAATAAGGGAGGAAGA NTRK2 NM_006180 15 381GCTCTCAGGACTGCAGAAGTACA GAGGAACCAATCCCACTCACAC NTRK2 NM_006180 16 383TCACTCTTTGCCTTCTGTCTCTG GCACTGTGCTTTGCTTTCTCAG NTRK3 NM_002530 17 385TGTCTCCTTTATCGTAGGTCTCCA CACCACATTTCCTACAGTTCCA NTRK3 NM_002530 18 387CACTGTGCACCAGACAGACAAA TGTGGTTTTCTGTATCAGCAGCTT PDGFRA NM_006206 18 389CAGGGAGTCTGAAATCATCAGG TCAAGTATCTAGCCCCAAATCCA PDGFRA NM_006206 19 391GGCAATATTGACCATTCATCATTC AGGCCAGGAGTAAGACGCAAC PDGFRB NM_002609 18 393AAGAACGTACGTGTGGTGTTGG CGCTATACTTGCTCTCCATGCACT PDGFRB NM_002609 19 395AGGAAACAGCCTCTGGTCCTC GTCAATGCTCAGACAGGGAGAT PTK7 NM_002821 18 397CCCAGGAAGGCAGGTACTGTTA TTTTACAACCACCAAGGGTGTG PTK7 NM_002821 19 399TCGTGTGGTTACCTCCAGATTTT AAATTAGCCAGGGAGTGGAGGT RET NM_000323 15 401CATGCCATGCTATGGCTCAC AGGCTGAGCGGAGTTCTAATTG RET NM_000323 16 403ATCTCAGCAATCCACAGGAGGT ATTTGCCTCACGAACACATCAT ROR1 NM_005012 09_1 405TGGAAAGTTGTCTATGGCACCTC ATGGGCAGCAAGGACTTACTCT ROR1 NM_005012 09_2 407CACCCCAATATTGTCTGCCTTC GGCTCGGGAACATGTAATTAGG ROR1 NM_005012 09_3 409CCATCATGTATGGCAAATTCTCTT TGGCGTCTCCTAGTAAAGATGCT ROR1 NM_005012 09_4 411GCCAGATTGCTGGTTTCATTG GGCTAAAACACAAAGCACCATT RYK NM_002958 13 413GGGAAGTCATCCACAAAGACCT GGTCTGGGTCACAGCTCCTC TEK NM_000459 18 415TTCTTCTGCCAAGATGTGGTGT TGCAGATGCTGCAATCATGTTA TEK NM_000459 19 427TGGACCGCGAAAGATAAATAGG TTCTGCTCCTCTGGAAACTG TIE NM_005424 18 419GGGTGAGAGCCAACACTGATCT CTGTGCCCTCTCATCTCACACT TI NM_005424 19 421AGAACCTAGCCTCCAAGATTGC ACACCTTCCAAGACTCCTTCCA TYRO3 NM_006293 18423, 424 GACTCGAGGGTGGGAGACAG GCTGTCACTAGGTGTCCTGAGC

TABLE S2 EGFR mutation status in untreated lung cancer Sequence SEQ IDSample Histology Source Gender Exon alteration NO Nucloetide Amino acidS0514 adenocarcinoma U.S. F 18 Substitution 425 2400G > A G719S S0377adenocarcinoma Japan F 18 Substitution 426 2400G > A G719S S0418adenocarcinoma Japan F 19 Del-1a 427 2481-2495delGGAATTAAGAGAAGCE746_A750del S0363 large cell ca. Japan F 19 Del-1a 4282481-2495delGGAATTAAGAGAAGC E746_A750del S0380 adenocarcinoma Japan M 19Del-1a 429 2481-2495delGGAATTAAGAGAAGC E746_A750del S0399 adenocarcinomaJapan F 19 Del-1a 430 2481-2495delGGAATTAAGAGAAGC E746_A750del S0353adenocarcinoma Japan F 19 Del-1a 431 2481-2495delGGAATTAAGAGAAGCE746_A750del S0385 adenocarcinoma Japan M 19 Del-1a 4322481-2495delGGAATTAAGAGAAGC E746_A750del S0301 adenocarcinoma Japan M 19Del-1a 433 2481-2495delGGAATTAAGAGAAGC E746_A750del S0412 adenocarcinomaJapan M 19 Del-1b 434 2482-2496delGGAATTAAGAGAAGC E746_A750del S0335adenocarcinoma Japan M 19 Del-1b 435 2482-2496delGGAATTAAGAGAAGCE746_A750del S0405 adenocarcinoma Japan F 19 Del-1b 4362482-2496delGGAATTAAGAGAAGC E746_A750del S0439 adenocarcinoma Japan M 19Del-2 437 2500-2523delTCTCCGAAGCCAACAAG S752_I759del GAAATC S0361adenocarcinoma Japan F 21 Substitution 438 2818T > G L858R S0388adenocarcinoma Japan F 21 Substitution 439 2818T > G L858R S0399adenocarcinoma Japan F 21 Substitution 440 2818T > G L858R References tonucleotide numbers correspond to SEQ ID NO: 511.

TABLE S3A EGFR mutation status in gefitinib-treated lung cancer SEQGefitinib Sequence ID sensitivity Sample Histology Source Gender Exonsalteration NO Nucleotide Amino acid Y IRIT adenocarcinoma U.S. M 19Del-3 441 2485-2493delTTAAGA L747_E749del, GAA, 2484G > C A750P Y P003adenocarcinoma U.S. M 19 Del-3 442 2485-2493delTTAAGA L747_E749del,GAA, 2484G > C A750P Y IR4T bronchioloalveolar U.S. F 19 Del-4 4432486-2503delTAAGAG L747_S752del, carcinoma AAGCAACATCTC P753S Y IR2Tadenocarcinoma U.S. F 19 Del-5 444 2484-2501delATTAAG L747_S752del,AGAAGCAACATC, E746V 2483A > T Y IR3T adenocarcinoma U.S. F 21Substitution 445 2818T > G L858R Y IRG adenocarcinoma U.S. F 21Substitution 446 2818T > G L858R in vitro H3255 N IR5 adenocarcinomaU.S. F 18-24 None detected n/a n/a N IR6 adenocarcinoma U.S. M 18-24None detected n/a n/a N IR8 adenocarcinoma U.S. F 18-24 None detectedn/a n/a N IR9 NSCLC U.S. F 18-24 None detected n/a n/a References tonucleotide numbers correspond to SEQ ID NO: 511.

TABLE S3B EGFR mutations not shown in Table 2, Table S2, or Table S3ASequence Seq. Sample Tissue Exon alteration Nucleotide Amino acidId. No. Tar4T Lung 19 Deletion 2485-2496delTTAAGAGAAGCA; L747_A750del;554 adenocarcinoma 2497A > C T751T AD355 Lung 19 Deletion2486-2496delTAAGAGAAGCA L747_T751del 720 adenocarcinoma IR TT Lung 19Deletion 2503-2517delCCGAAAGCCAACAAG P753_K757del 721 adenocarcinomaAD240 Lung 20 Insertion 2555-2556insCAACCCGG D770_N771insNPG 722adenocarcinoma AD261 Lung 20 Insertion 2557-2558insGCGTGGACAD770_N771insSVD 723 adenocarcinoma Lung 20 Insertion 2562-2563insGGTP772_H773insV 724 adenocarcinoma AD356 Lung 20 Substitution2580-2581GG > AA G779S 725 adenocarcinoma SP02-23 Acute myeloid 21Substitution 2816G > T G857V 726 leukemia SP08-94 Glioma 21 Substitution2827T > A L861Q 727 SP06-45 Sarcoma 21 Substitution 2894T > C L883S 728AD241 Colon 22 Substitution 2932G > T D896Y 729 adenocarcinoma

TABLE S3C Position of BCR-ABL mutants resistant to imatinib andanalogous positions in EGFR Abl1 residue subject to AnalogousIdentical/similar/ resistance mutation EGFR residue non-conservedMet-244 Lys-714 Non-conserved Leu-248 Leu-718 Identical Gly-250 Ser-720Non-conserved Gln-252 Ala-722 Non-conserved Tyr-253 Phe-723 SimilarGlu-255 Thr-725 Non-conserved Asp-276 Ala-750 Non-conservd Thr-315Thr-790 Identical Phe-317 Leu-792 Similar Met-351 Met-825 IdenticalGlu-355 Glu-829 Identical Phe-359 Leu-833 Similar His-396 His-870Identical Ser-417 Thr-892 Similar Phe-486 Phe-961 Identical

TABLE S4 Primers used for cDNA sequencing Primer name SEQ ID NOPrimer sequence 5′ to 3′ cDNA_EGFR_aF 447TGTAAAACGACGGCCAGTCGCCCAGACCGGACGACA cDNA_EGFR_aR 448CAGGAAACAGCTATGACCAGGGCAATGAGGACATAACCA cDNA_EGFR_bF 449TGTAAAACGACGGCCAGTGGTGGTCCTTGGGAATTTGG cDNA_EGFR_bR 450CAGGAAACAGCTATGACCCCATCGACATGTTGCTGAGAAA cDNA_EGFR_cF 451TGTAAAACGACGGCCAGTGAAGGAGCTGCCCATGAGAA cDNA_EGFR_cR 452CAGGAAACAGCTATGACCCGTGGCTTCGTCTCGGAATT cDNA_EGFR_dF 453TGTAAAACGACGGCCAGTGAAACTGACCAAAATCATCTGT cDNA_EGFR_dR 454CAGGAAACAGCTATGACCTACCTATTCCGTTACACACTTT cDNA_EGFR_eF 455TGTAAAACGACGGCCAGTCCGTAATTATGTGGTGACAGAT cDNA_EGFR_eR 456CAGGAAACAGCTATGACCGCGTATGATTTCTAGGTTCTCA cDNA_EGFR_fF 457TGTAAAACGACGGCCAGTCTGAAAACCGTAAAGGAAATCAC cDNA_EGFR_fR 458CAGGAAACAGCTATGACCCCTGCCTCGGCTGACATTC cDNA_EGFR_gF 459TGTAAAACGACGGCCAGTTAAGCAACAGAGGTGAAAACAG cDNA_EGFR_gR 460CAGGAAACAGCTATGACCGGTGTTGTTTTCTCCCATGACT cDNA_EGFR_hF 461TGTAAAACGACGGCCAGTGGACCAGACAACTGTATCCA cDNA_EGFR_hR 462CAGGAAACAGCTATGACCTTCCTTCAAGATCCTCAAGAGA cDNA_EGFR_iF 463TGTAAAACGACGGCCAGTGATCGGCCTCTTCATGCGAA cDNA_EGFR_iR 464CAGGAAACAGCTATGACCACGGTGGAGGTGAGGCAGAT cDNA_EGFR_jF 465TGTAAAACGACGGCCAGTCGAAAGCCAACAAGGAAATCC cDNA_EGFR_jR 466CAGGAAACAGCTATGACCATTCCAATGCCATCCACTTGAT cDNA_EGFR_kF 467TGTAAAACGACGGCCAGTAACACCGCAGCATGTCAAGAT cDNA_EGFR_kR 468CAGGAAACAGCTATGACCCTCGGGCCATTTTGGAGAATT cDNA_EGFR_IF 469TGTAAAACGACGGCCAGTTCAGCCACCCATATGTACCAT cDNA_EGFR_IR 470CAGGAAACAGCTATGACCGCTTTGCAGCCCATTTCTATC cDNA_EGFR_mF 471TGTAAAACGACGGCCAGTACAGCAGGGCTTCTTCAGCA cDNA_EGFR_mR 472CAGGAAACAGCTATGACCTGACACAGGTGGGCTGGACA cDNA_EGFR_nF 473TGTAAAACGACGGCCAGTGAATCCTGTCTATCACAATCAG cDNA_EGFR_nR 474CAGGAAACAGCTATGACCGGTATCGAAAGAGTCTGGATTT cDNA_EGFR_oF 475TGTAAAACGACGGCCAGTGCTCCACAGCTGAAAATGCA cDNA_EGFR_oR 476CAGGAAACAGCTATGACCACGTTGCAAAACCAGTCTGTG

1. A method to determine an increased likelihood of response totreatment with gefitinib or erlotinib in an individual affected withnon-small cell lung cancer (NSCLC) comprising: determining by polymerasechain reaction, the presence or absence of at least one nucleotidevariance in exon 18, 19, or 21 of the epidermal growth factor receptor(EGFR) gene in DNA obtained from an NSCLC sample from said individual,wherein said at least one nucleotide variance is selected from: 1) anin-frame deletion in exon 19 of the EGFR gene comprising a deletionwithin codons 746 to 753 that results in amino acid changes consistingof a deletion of at least amino acids leucine, arginine, and glutamicacid at position 747, 748, and 749 of SEQ ID NO:512; 2) a substitutionin exon 21 that results in an amino acid change comprising asubstitution of arginine for leucine at position 858 (L858R) of SEQ IDNO:512, or a substitution in exon 21 that results in an amino acidchange consisting of a substitution of glutamine for leucine at position861 (L861Q) of SEQ ID NO:512; or 3) a substitution in exon 18 thatresults in an amino acid change comprising substitution of cysteine forglycine at position 719 (G719C) of SEQ ID NO:512; wherein the presenceof said at least one nucleotide variance indicates that said individualis likely to respond to treatment with gefitinib or erlotinib.
 2. Themethod of claim 1 wherein said at least one nucleotide variance is anin-frame deletion in exon 19 within codons 746 to 753 that results inamino acid changes comprising a deletion of at least amino acidsleucine, arginine, and glutamic acid at position 747, 748, and 749 ofSEQ ID NO:512.
 3. The method of claim 1, wherein said at least onenucleotide variance is a substitution in exon 21 that results in asubstitution of arginine for leucine at position 858 (L858R) of SEQ IDNO:512 or a substitution of glutamine for leucine at position 861(L861Q) of SEQ ID NO:512.
 4. The method of claim 1, wherein said atleast one nucleotide variance is a substitution in exon 18 that resultsin a substitution of cysteine for glycine at position 719 (G719C) of SEQID NO:512.